Fc variants that extend antibody half-life

ABSTRACT

The invention relates generally to compositions and methods for altering the serum half-life in vivo of an antibody.

This application claims the benefit under 35 U.S.C. 119 to U.S.Provisional Application Ser. No. 61/392,115, filed Oct. 12, 2010, and isalso a continuation-in-part of U.S. Ser. No. 12/341,769, filed Dec. 22,2008, which are both incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present application relates to optimized IgG immunoglobulin variantsthat extend antibody half-life in vivo, and their application,particularly for therapeutic purposes.

BACKGROUND OF THE INVENTION

Antibodies are immunological proteins that bind a specific antigen. Inmost mammals, including humans and mice, antibodies are constructed frompaired heavy and light polypeptide chains. Each chain is made up ofindividual immunoglobulin (Ig) domains, and thus the generic termimmunoglobulin is used for such proteins. Each chain is made up of twodistinct regions, referred to as the variable and constant regions. Thelight and heavy chain variable regions show significant sequencediversity between antibodies, and are responsible for binding the targetantigen. The constant regions show less sequence diversity, and areresponsible for binding a number of natural proteins to elicit importantbiochemical events. In humans there are five different classes ofantibodies including IgA (which includes subclasses IgA1 and IgA2), IgD,IgE, IgG (which includes subclasses IgG1, IgG2, IgG3, and IgG4), andIgM. The distinguishing feature between these antibody classes is theirconstant regions, although subtler differences may exist in the Vregion, IgG antibodies are tetrameric proteins composed of two heavychains and two light chains. The IgG heavy chain is composed of fourimmunoglobulin domains linked from N- to C-terminus in the orderVH—CH1-CH2-CH3, referring to the heavy chain variable domain, heavychain constant domain 1, heavy chain constant domain 2, and heavy chainconstant domain 3 respectively (also referred to as VH—Cγ1-Cγ2-Cγ3,referring to the heavy chain variable domain, constant gamma 1 domain,constant gamma 2 domain, and constant gamma 3 domain respectively). TheIgG light chain is composed of two immunoglobulin domains linked from N-to C-terminus in the order VL-CL, referring to the light drain variabledomain and the light chain constant domain respectively.

In IgG, a site on Fc between the Cγ2 and Cγ3 domains mediatesinteraction with the neonatal receptor FcRn. Binding to FcRn recyclesendocytosed antibody from the endosome back to the bloodstream (Raghavanet al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ghetie et al., 2000,Annu Rev Immunol 18:739-766, both entirely incorporated by reference).This process, coupled with preclusion of kidney filtration due to thelarge size of the full-length molecule, results in favorable antibodyserum half-lives ranging from one to three weeks. Binding of Fc to FcRnalso plays a key role in antibody transport. The binding site on Fc forFcRn is also the site at which the bacterial proteins A and G bind. Thetight binding by these proteins is typically exploited as a means topurify antibodies by employing protein A or protein G affinitychromatography during protein purification. Thus the fidelity of thisregion on Fc is important for both the clinical properties of antibodiesand their purification. Available structures of the rat Fc/FcRn complex(Burmeister et al., 1994, Nature, 372:379-383; Martin et al., 2001, MolCell 7:867-877, both entirely incorporated by reference), and of thecomplexes of Fc with proteins A and G (Deisenhofer, 1981, Biochemistry20:2361-2370; Sauer-Eriksson et al., 1995, Structure 3:265-278; Tashiroet al., 1995, Curr Opin Struct Biol 5:471-481, all entirely incorporatedby reference), provide insight into the interaction of Fc with theseproteins. The FcRn receptor is also responsible for the transfer of IgGto the neonatal gut and to the lumen of the intestinal epithelia inadults (Ghetie and Ward, Annu. Rev. Immunol, 2000, 18:739-766; Yoshidaet al., Immunity, 2004, 20(6):769-783, both entirely incorporated byreference).

Antibodies have serum half-lives in vivo ranging from one to threeweeks. This favorable property is due to the preclusion of kidneyfiltration due to the large size of the full-length molecule, andinteraction of the antibody Fc region with the neonatal Fc receptorFcRn. Binding to FcRn recycles endocytosed antibody from the endosomeback to the bloodstream (Raghavan et al., 1996, Annu Rev Cell Dev Biol12:181-220; Ghetie et al., 2000, Annu Rev Immunol 18:739-766, bothentirely incorporated by reference).

Other properties of the antibody may determine its clearance rate (e.g.stability and half-life) in vivo. In addition to antibody binding to theFcRn receptor, other factors that contribute to clearance and half-lifeare serum aggregation, enzymatic degradation in the serum, inherentimmunogenicity of the antibody leading to clearing by the immune system,antigen-mediated uptake, FcR (non-FcRn) mediated uptake and non-serumdistribution (e.g. in different tissue compartments).

There is a need for antibody modifications that improve thepharmacokinetic properties of antibodies. The present application meetsthese and other needs and provides novel engineered variants in theconstant regions to improve serum half-life.

BRIEF SUMMARY OF THE INVENTION Problem to be Solved

Accordingly, one problem to be solved is to increase serum half life ofantibodies by altering the constant domains, thus allowing the sameconstant regions to be used with different antigen binding sequences,e.g. the variable regions including the CDRs, and minimizing thepossibility of immunogenic alterations. Thus providing antibodies withconstant region variants with extended half-life provides a modularapproach to improving the pharmacokinetic properties of antibodies, asdescribed herein. In addition, due to the methodologies outlined herein,the possibility of immunogenicity resulting from the FcRn variants issignificantly reduced by importing variants from different IgG isotypessuch that serum half-life is increased without introducing significantimmunogenicity.

SUMMARY

In one aspect, the present invention provides an antibody comprising avariant Fc region as compared to a parent Fc region, wherein the variantFc region comprises a first mutation which is a serine at position 434and a second mutation selected from the group: an isoleucine at position311, a valine at position 311, an isoleucine at position 436, and avaline at position 436. In a further aspect, the antibody has increasedserum half-life as compared to an antibody comprising the parent Fcregion, wherein numbering is according to the EU index. In a stillfurther aspect, the antibody has increased binding affinity to a humanFcRn receptor as compared to an antibody comprising the parent Fcregion, wherein numbering is according to the EU index.

In one embodiment, the present invention provides nucleic acids encodingany of the variant Fc regions described herein.

In a further embodiment, the present invention provides host cellscomprising a nucleic acid encoding any of the variant Fc regionsdescribed herein.

In one aspect, the present invention provides methods of administeringan antibody to a subject, where the antibody comprises a variant Fcregion as compared to a parent Fc region, wherein the variant Fc regioncomprises a first mutation which is a serine at position 434 and asecond mutation selected from the group: an isoleucine at position 311,a valine at position 311, an isoleucine at position 436, and a valine atposition 436, where the antibody has increased serum half-life ascompared to an antibody comprising the parent Fc region, and whereinnumbering is according to the EU index.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Sequence alignments of human IgG constant heavy chains. Grayindicates differences from IgG1, and boxed residues indicate commonallotypic variations in the human population.

FIG. 2. (SEQ ID NO: 1-6) Amino acid sequences of constant regions usedin the invention.

FIG. 3. (SEQ ID NO: 7-8) Amino acid sequences of exemplary variantconstant regions.

FIG. 4. (SEQ ID NO: 9-10) Amino acid sequences of VH and VL variableregions used in the invention.

FIG. 5. Relative VEGF binding by WT and select variant IgG1 anti-VEGFantibodies. The plot shows the Biacore response units (RU) at the end ofthe association phase for binding of antibody analyte to immobilizedVEGF antigen. Anti-Her2 IgG1 antibody was used as a negative control.

FIG. 6. Biacore sensorgrams of WT and variant IgG1 antibodies toimmobilized human FcRn at low (6.0) and high (7.4) pH.

FIG. 7. FcRn binding affinities of WT and select variant IgG1 antibodiesto human FcRn at pH 6.0 as determined by Biacore. The graph shows a plotof the pseudo-affinity constant (Ka*), on a log scale.

FIG. 8 a-8 b. In vivo pharmacokinetics of WT and variant antibodies inmFcRn−/− hFcRn+ mice. The graphs plot the serum concentration ofantibody versus time after a single intravenous dose. FIG. 8 a showsdata from one of the 4 studies carried out with IgG1 antibodies (Study3), and FIG. 8 b shows data from a study carried out with IgG2antibodies (Study 5).

FIG. 9. Fitted PK parameters from all in vivo PK studies carried out inmFcRn^(−/−) hFcRn⁺ mice with variant and WT antibodies, n represents thenumber of mice per group, with Mean and standard deviation (SD) dataprovided for PK parameters. Half-Life represents the beta phase thatcharacterizes elimination of antibody from serum. Cmax is the maximalobserved serum concentration, AUG is the area under the concetrationtime curve, and clearance is the clearance of antibody from serum. Foldhalf-life is calculated as the half-life of variant antibody over thatof the WT IgG1 or IgG2 parent within each study.

FIG. 10 a-10 b. Relative binding of variant IgG1 anti-VEGF antibodies tocynomolgus monkey and human FcRn as determined by Biacore. FIG. 10 ashows the data in tabular form. FIG. 10 b shows a plot of the data.

FIG. 11. In vivo pharmacokinetics of WT and variant antibodies incynomolgus monkeys. The graphs plot the serum concentration of antibodyversus time after a single intravenous dose.

FIG. 12. Fitted PK parameters from the in vivo PK study in cynomolgusmonkeys with variant and WT antibodies. Parameters are as described inFIG. 9.

FIG. 13. Designed Fc variants. Variants are screened using the describedmethods in order to obtain modifications that extend the in vivohalf-life of antibodies.

FIG. 14. Screen of engineered Fc variants for binding to human FcRn. Thetable shows the off-rate (k_(off)) for binding of each variant to humanFcRn at pH 6.0 by Biacore.

FIG. 15. Graph of k_(off) for screened Fc variants (data plotted arefrom FIG. 14).

FIG. 16. Affinities of select variants for human FcRn at pH 6.0 based ona FcRn concentration series Biacore experiment. The data show the on andoff kinetic rate constants (k_(on) and k_(off) respectively), and theassociation and dissociation equilibrium constants (K_(A) and K_(D)respectively), as well as the fold improvement in K_(D) and k_(off)relative to WT IgG1 and IgG1/2N434S.

FIG. 17. Plot off affinities of select variants for human FcRn at pH 6.0as determined by Biacore. Values are plotted from FIG. 16.

FIG. 18. Plot off affinities of select variants for human FcRn at pH 6.0as determined by Biacore. Values are plotted from FIG. 16.

FIG. 19. Designed single variants

FIG. 20. Designed combination variants

FIG. 21. Affinities of select variants for human FcRn at pH 6.0 based ona FcRn concentration series Biacore experiment. The data show thedissociation equilibrium constant KD, as well as the fold improvement inKD relative to the IgG1/2 parent.

FIG. 22. Plot off affinities of select variants for human FcRn at pH 6.0as determined by Biacore. Values are plotted from FIG. 21.

FIG. 23. In vivo pharmacokinetics of IgG1/2 variant antibodies inmFcRn−/− hFcRn+ mice. The graphs plot the serum concentration ofantibody versus time after a single intravenous dose.

FIG. 24. Fitted half-lives (t½) from the PK study in hFcRn⁺ mice,n=number of mice, columns n1-n5 show the half-lives of the individualmice in each group, and the average half-life and standard deviation areshown in the last two columns.

FIG. 25. Scatter plot of half-life results from PK study in hFcRn⁺ mice.Data are plotted from FIG. 24. Each dot represents the t½ of anindividual mouse within each variant group, and the line indicates theaverage.

DETAILED DESCRIPTION OF THE INVENTION I. Overview

The present invention discloses the generation of novel variants of Fcdomains, including those found in antibodies, Fc fusions, andimmuno-adhesions, which have an increased binding to the FcRn receptor.As noted herein, binding to FcRn results in longer serum retention invivo. These variants of Fc domains are also referred to herein as “Fcvariants” or “FcRn variants”.

The substitutions in the Fc domains (“Fc substitutions”) are chosen suchthat the resultant proteins (“Fc proteins”) show improved serumhalf-life in vivo as compared to the wild type protein. In order toincrease the retention of the Fc proteins in vivo, the increase inbinding affinity must be at around pH 6 while maintaining lower affinityat around pH 7.4. Although still under examination, Fc regions arebelieved to have longer half-lives in vivo, because binding to FcRn atpH 6 in an endosome sequesters the Fc (Ghetie and Ward, 1997 ImmunolToday. 18(12): 592-598, entirely incorporated by reference). Theendosomal compartment then recycles the Fc to the cell surface. Once thecompartment opens to the extracellular space, the higher pH, ˜7.4,induces the release of Fc back into the blood. In mice, Dall' Acqua etal. showed that Fc mutants with increased FcRn binding at pH 6 and pH7.4 actually had reduced serum concentrations and the same half life aswild-type Fc (Dall' Acqua et al. 2002, J. Immunol. 169:5171-5180,entirely incorporated by reference). The increased affinity of Fc forFcRn at pH 7.4 is thought to forbid the release of the Fc back into theblood. Therefore, the Fc mutations that will increase Fc's half-life invivo will ideally increase FcRn binding at the lower pH while stillallowing release of Fc at higher pH. The amino acid histidine changesits charge state in the pH range of 6.0 to 7.4. Therefore, it is notsurprising to find His residues at important positions in the Fc/FcRncomplex.

An additional aspect of the invention is the increase in FcRn bindingover wild type specifically at lower pH, about pH 6.0, to facilitateFc/FcRn binding in the endosome. Also disclosed are Fc variants withaltered FcRn binding and altered binding to another class of Fcreceptors, the FcγR's (sometimes written FcgammaR's) as differentialbinding to FcγRs, particularly increased binding to FcγRIIIb anddecreased binding to FcγRIIb, has been shown to result in increasedefficacy.

In addition, many embodiments of the invention rely on the “importation”of substitutions at particular positions from one IgG isotype intoanother, thus reducing or eliminating the possibility of unwantedimmunogenicity being introduced into the variants. That is, IgG1 is acommon isotype for therapeutic antibodies for a variety of reasons,including high effector function. IgG2 residues at particular positionscan be introduced into the IgG1 backbone to result in a protein thatexhibits longer serum half-life.

In other embodiments, non-isotypic amino acid changes are made, toimprove binding to FcRn and/or to increase in vivo serum halt-life,and/or to allow accommodations in structure for stability, etc. as ismore further described below.

As will be appreciated by those in the art and described below, a numberof factors contribute to the in vivo clearance, and thus the half-life,of antibodies in serum. One factor involves the antigen to which theantibody binds; that is, antibodies with identical constant regions butdifferent variable regions (e.g. Fv domains), may have differenthalf-lives due to differential ligand binding effects. However, thepresent invention demonstrates that while the absolute half life of twodifferent antibodies may differ due to these antigen specificityeffects, the FcRn variants described herein, can transfer to differentligands to give the same trends of increasing half-life. That is, ingeneral, the relative “order” of the FcRn binding/half life increaseswill track to antibodies with the same variants of antibodies withdifferent Fvs as is discussed herein.

II. Description of the Invention A. Antibodies

The present invention relates to the generation of Fc variants ofantibodies, generally therapeutic antibodies. As is discussed below, theterm “antibody” is used generally. Antibodies that find use in thepresent invention can take on a number of formats as described herein,including traditional antibodies as well as antibody derivatives,fragments and mimetics, described below. In general, the term “antibody”includes any polypeptide that includes at least one constant domain,including, but not limited to, CH1, CH2, CH3 and CL.

Traditional antibody structural units typically comprise a tetramer.Each tetramer is typically composed of two identical pairs ofpolypeptide chains, each pair having one “light” (typically having amolecular weight of about 25 kDa) and one “heavy” chain (typicallyhaving a molecular weight of about 50-70 kDa). Human light chains areclassified as kappa and lambda light chains. The present invention isdirected to the IgG class, which has several subclasses, including, butnot limited to IgG1, IgG2, IgG3, and IgG4. Thus, “isotype” as usedherein is meant any of the subclasses of immunoglobulins defined by thechemical and antigenic characteristics of their constant regions. Itshould be understood that therapeutic antibodies can also comprisehybrids of isotypes and/or subclasses. For example, as shown herein, thepresent invention covers Fc variant engineering of IgG1/G2 hybrids.

The amino-terminal portion of each chain includes a variable region ofabout 100 to 110 or more amino acids primarily responsible for antigenrecognition, generally referred to in the art and herein as the “Fvdomain” or “Fv region”. In the variable region, three loops are gatheredfor each of the V domains of the heavy chain and light chain to form anantigen-binding site. Each of the loops is referred to as acomplementarity-determining region (hereinafter referred to as a “CDR”),in which the variation in the amino acid sequence is most significant.“Variable” refers to the fact that certain segments of the variableregion differ extensively in sequence among antibodies. Variabilitywithin the variable region is not evenly distributed. Instead, the Vregions consist of relatively invariant stretches called frameworkregions (FRs) of 15-30 amino acids separated by shorter regions ofextreme variability called “hypervariable regions” that are each 9-15amino acids long or longer.

Each VH and VL is composed of three hypervariable regions(“complementary determining regions,” “CDRs”) and four FRs, arrangedfrom amino-terminus to carboxy-terminus in the following order:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

The hypervariable region generally encompasses amino acid residues fromabout amino acid residues 24-34 (LCDR1; “L” denotes light chain), 50-56(LCDR2) and 89-97 (LCDR3) in the light chain variable region and aroundabout 31-35B (HCDR1; “H” denotes heavy chain), 50-65 (HCDR2), and 95-102(HCDR3) in the heavy chain variable region; Kabat et al., SEQUENCES OFPROTEINS OF IMMUNOLOGICAL INTEREST, 5^(th) Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991) and/or thoseresidues forming a hypervariable loop (e.g. residues 26-32 (LCDR1),50-52 (LCDR2) and 91-96 (LCDR3) in the light chain variable region and26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chainvariable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917.Specific CDRs of the invention are described below.

Throughout the present specification, the Kabat numbering system isgenerally used when referring to a residue in the variable domain(approximately, residues 1-107 of the light chain variable region andresidues 1-113 of the heavy chain variable region) (e.g, Kabat et al.,supra (199.1)).

The CDRs contribute to the formation of the antigen-binding, or morespecifically, epitope binding site of antibodies. “Epitope” refers to adeterminant that interacts with a specific antigen binding site in thevariable region of an antibody molecule known as a paratope. Epitopesare groupings of molecules such as amino acids or sugar side chains andusually have specific structural characteristics, as well as specificcharge characteristics. A single antigen may have more than one epitope.

The epitope may comprise amino acid residues directly involved in thebinding (also called immunodominant component of the epitope) and otheramino acid residues, which are not directly involved in the binding,such as amino acid residues which are effectively blocked by thespecifically antigen binding peptide; in other words, the amino acidresidue is within the footprint of the specifically antigen bindingpeptide.

Epitopes may be either conformational or linear. A conformationalepitope is produced by spatially juxtaposed amino acids from differentsegments of the linear polypeptide chain. A linear epitope is oneproduced by adjacent amino acid residues in a polypeptide chain.Conformational and nonconformational epitopes may be distinguished inthat the binding to the former but not the latter is lost in thepresence of denaturing solvents.

An epitope typically includes at least 3, and more usually, at least 5or 8-10 amino acids in a unique spatial conformation. Antibodies thatrecognize the same epitope can be verified in a simple immunoassayshowing the ability of one antibody to block the binding of anotherantibody to a target antigen, for example “binning.”

As will be appreciated by those in the art, a wide variant of antigenbinding domains, e.g. Fv regions, may find use in the present invention.Virtually any antigen may be targeted by the IgG variants, including butnot limited to proteins, subunits, domains, motifs, and/or epitopesbelonging to the following list of target antigens, which includes bothsoluble factors such as cytokines and membrane-bound factors, includingtransmembrane receptors: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a,8-oxo-dG, A1 Adenosine Receptor, A33, ACE, ACE-2, Activin, Activin A,Activin AB, Activin B, Activin C, Activin RIA, Activin RIA ALK-2,Activin RIB ALK-4, Activin RIIA, Activin RIIB, ADAM, ADAM10, ADAM12,ADAM15, ADAM17/TACE, ADAMS, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, Addressins,aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V/beta-1antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART,Artemin, anti-Id, ASPARTIC, Atrial natriuretic factor, av/b3 integrin,Ax1, b2M, B7-1, B7-2, B7-H, B-lymphocyte Stimulator (BlyS), BACE,BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA,BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a,BMP-3 Osteogenin, BMP-4 BMP-2b, BMP-5, BMP-6 Vgr-1, BMP-7 (OP-1), BMP-8(BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1,BMPR-II (BRK-3), BMPs, b-NGF, BOK, Bombesin, Bone-derived neurotrophicfactor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a,C10, CA125, CAD-8, Calcitonin, cAMP, carcinoembryonic antigen (CEA),carcinoma-associated antigen, Cathepsin A, Cathepsin B, CathepsinC/DPPI, Cathepsin D, Cathepsin E, Cathepsin H, Cathepsin L, Cathepsin O,Cathepsin S, Cathepsin V, Cathepsin X/Z/P, CBL, CCI, CCK2, CCL, CCL1,CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2,CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3,CCL4. CCL5, CCL6, CCL7, CCL8. CCL9/10, CCR, CCR1, CCR10, CCR10, CCR2,CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5,CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13 CD14, CD15, CD16, CD18,CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L,CD32, CD33 (p67 proteins), CD34, CD38, CD40, CD40L, CD44, CD45, CD46,CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1),CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152,CD164, CEACAM5, CFTR, cGMP, CINC, Clostridium botulinum toxin,Clostridium perfringens toxin, CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1,COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL,CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10,CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2,CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN,DCC, DcR3, DC-SIGN, Decay accelerating factor, des(1-3)-IGF-I (brainIGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV/CD26, Dtk, ECAD, EDA,EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelinreceptor, Enkephalinase, eNOS, Eot, eotaxin1, EpCAM, Ephrin B2/EphB4,EPO, ERCC, E-selectin, ET-1, Factor IIa, Factor VII, Factor VIIIc,Factor IX, fibroblast activation protein (FAP), Fas, FcR1, FEN-1,Ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, Fibrin, FL,FLIP, Flt-3, Flt-4, Follicle stimulating hormone, Fractalkine, FZD1,FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas 6,GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14,CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8(Myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1,GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, Glucagon, Glut 4, glycoproteinIIb/IIIa (GP IIb/IIIa), GM-CSF, gp130, gp72, GRO, Growth hormonereleasing factor, Hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gBenvelope glycoprotein, HCMV) gH envelope glycoprotein, HCMV UL,Hemopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2/neu(ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gBglycoprotein, HSV gD glycoprotein, HGFA, High molecular weightmelanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp120 V3loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin,human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, 1-309,IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF,IGF binding proteins, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R,IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10,IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-alpha,INF-beta, INF-gamma, Inhibin, iNOS, Insulin A-chain, Insulin B-chain,Insulin-like growth factor 1, integrin alpha2, integrin alpha3, integrinalpha4, integrin alpha4/beta1, integrin alpha4/beta7, integrin alpha5(alphaV), integrin alpha5/beta1, integrin alpha5/beta3, integrin alpha6,integrin beta1, integrin beta2, interferon gamma, IP-10, I-TAC, JE,Kallikrein 2, Kallikrein 5, Kallikrein 6, Kallikrein 11, Kallikrein 12,Kallikrein 14, Kallikrein 15, Kallikrein L1, Kallikrein L2, KallikreinL3, Kallikrein L4, KC, KDR, Keratinocyte Growth Factor (KGF), laminin 5,LAMP, LAP, LAP (TGF-1), Latent TGF-1, Latent TGF-1 bp1, LBP, LDGF,LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3,Lfo, LIF, LIGHT, lipoproteins, LIX, LKN, Lptn, L-Selectin, LT-a, LT-b,LTB4, LTBP-1, Lung surfactant, Luteinizing hormone, Lymphotoxin BetaReceptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF,MDC, Mer, METALLOPROTEASES, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG,MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13,MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo,MSK, MSP, mucin (Muc1), MUC18, Muellerian-inhibitin substance, Mug,MuSK, NAIP, NAP, NCAD, N-Cadherin, NCA 90, NCAM, NCAM, Neprilysin,Neurotrophin-3, -4, or -6, Neurturin, Neuronal growth factor (NGF),NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN,OSM, OX40L, OX40R, p150, p95, PADPr, Parathyroid hormone, PARC, PARP,PBR, PBSF, PCAD, P-Cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4,PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (FLAP),PlGF, PLP, PP14, Proinsulin, Prorelaxin, Protein C, PS, PSA, PSCA,prostate specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51,RANK, RANKL, RANTES, RANTES, Relaxin A-chain, Relaxin B-chain, renin,respiratory syncytial virus (RSV) F, RSV Fgp, Ret, Rheumatoid factors,RLIP76, RPA2, RSK, S100, SCF/KL, SDF-1, SERINE, Serum albumin, sFRP-3,Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat,STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72),TARC, TCA-3, T-cell receptors (e.g., T-cell receptor alpha/beta), TdT,TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkalinephosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan Specific,TGF-beta R1 (ALK-5), TGF-beta RII, TGF-beta RIIb, TGF-beta RIII,TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, Thrombin, ThymusCk-1, Thyroid stimulating hormone, Tie, TIMP, TIQ, Tissue Factor,TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-beta2, TNFα,TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSF10B (TRAIL R2DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID),TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R),TNFRSF11B (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACI),TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16(NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROYTAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF RI CD120a, p55-60),TNFRSF1B (TNF RII CD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNFRUT, TNFC R), TNFRSF4 (OX40 ACT35, TXGP1 R), TNFRSF5 (CD40 p50), TNFRSF6(Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3 M68, TR6), TNFRSF7 (CD27),TNFRSF8 (CD30), TNFRSF9 (4-1BB CD137, ILA), TNFRSF21 (DR6), TNFRSF22(DcTRAIL R2 TNFRH2), TNFRST23 (DcTRAIL R1 TNFRH1), TNFRSF25 (DR3 Apo-3,LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 Ligand, TL2), TNFSF11(TRANCE/RANK Ligand ODF, OPG Ligand), TNFSF12 (TWEAK Apo-3 Ligand, DR3Ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL 1, THANK,TNFSF20), TNFSF14 (LIGHT HVEM Ligand, LTg), TNFSF15 (TL1A/VEGI), TNFSF18(GITR Ligand AITR Ligand, TL6), TNFSF1A (TNF-a Conectin, DIF, TNFSF2),TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 Ligandgp34, TXGP1), TNFSF5 (CD40 Ligand CD154, gp39, HIGM1, IMD3, TRAP),TNFSF6 (Fas Ligand Apo-1 Ligand, APT1 Ligand), TNFSF7 (CD27 LigandCD70), TNFSF8 (CD30 Ligand CD153), TNFSF9 (4-1BB Ligand CD137 Ligand),TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE,transferring receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associatedantigen CA 125, tumor-associated antigen expressing Lewis Y relatedcarbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, Urokinase, VCAM, VCAM-1,VECAD, VE-Cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3(flt-4), VEGI VIM, Viral antigens, VLA, VLA-1, VLA-4, VNR integrin, vonWillebrands factor, WIF-1, WNT1, WNT2, WNT2B/13, WNT3, WNT3A, WNT4,WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B,WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD,and receptors for hormones and growth factors.

In some embodiments, the engineering of Fc domains described herein isdone to therapeutic antibodies. A number of antibodies that are approvedfor use, in clinical trials, or in development may benefit from the Fcvariants of the present invention. These antibodies are herein referredto as “clinical products and candidates”. Thus in a preferredembodiment, the engineered constant region(s) of the present inventionmay find use in a range of clinical products and candidates. Forexample, a number of antibodies that target CD20 may benefit from the Fcengineering of the present invention. For example the Fc variants of thepresent invention may find use in an antibody that is substantiallysimilar to rituximab (Rituxan®, IDEC/Genentech/Roche) (see for exampleU.S. Pat. No. 5,736,137), a chimeric anti-CD20 antibody approved totreat Non-Hodgkin's lymphoma; HuMax-CD20, an anti-CD20 currently beingdeveloped by Genmab, an anti-CD20 antibody described in U.S. Pat. No.5,500,362, AME-133 (Applied Molecular Evolution), hA20 (Immunomedics,Inc.), HumaLYM (Intracel), and PRO70769 (PCT/US2003/040426, entitled“Immunoglobulin Variants and Uses Thereof”). A number of antibodies thattarget members of the family of epidermal growth factor receptors,including EGFR (ErbB-1), Her2/neu (ErbB-2), Her3 (ErbB-3), Her4(ErbB-4), may benefit from Fc engineered constant region(s) of theinvention. For example the Fc engineered constant region(s) of theinvention may find use in an antibody that is substantially similar totrastuzumab (Herceptin®, Genentech) (see for example U.S. Pat. No.5,677,171), a humanized anti-Her2/neu antibody approved to treat breastcancer; pertuzumab (rhuMab-2C4, Omnitarg™), currently being developed byGenentech; an anti-Her2 antibody described in U.S. Pat. No. 4,753,894;cetuximab (Erbitux®, Imclone) (U.S. Pat. No. 4,943,533; PCT WO96/40210), a chimeric anti-EGFR antibody in clinical trials for avariety of cancers; ABX-EGF (U.S. Pat. No. 6,235,883), currently beingdeveloped by Abgenix-Immimex-Amgen; HuMax-EGFr (U.S. Ser. No.10/172,317), currently being developed by Genmab; 425, EMD55900,EMD62000, and EMD72000 (Merck KGaA) (U.S. Pat. No. 5,558,864; Murthy etal. 1987, Arch Biochem Biophys. 252(2):549-60; Rodeck et al., 1987, JCell Biochem. 35(4):315-20; Kettleborough et al., 1991, Protein Eng.4(7):773-83); ICR62 (Institute of Cancer Research) (PCT WO 95/20045;Modjtahedi et al., 1993, J. Cell Biophys. 1993, 22(1-3): 129-46;Modjtahedi et al., 1993, Br J Cancer. 1993, 67(2):247-53; Modjtahedi etal, 1996, Br J Cancer, 73(2):228-35; Modjtahedi et al, 2003, Int JCancer, 105(2):273-80); TheraCIM hR3 (YM Biosciences, Canada and Centrode Immunologia Molecular, Cuba (U.S. Pat. No. 5,891,996; U.S. Pat. No.6,506,883; Mateo et al, 1997, Immunotechnology, 3(1):71-81); mAb-806(Ludwig Institute for Cancer Research, Memorial Sloan-Kettering)(Jungbluth et al. 2003, Proc Natl Acad Sci USA. 100(2):639-44); KSB-102(KS Biomedix); MR1-1 (IVAX, National Cancer Institute) (PCT WO0162931A2); and SC100 (Scancell) (PCT WO 01/88138). In another preferredembodiment, the Fc engineered constant region(s) of the presentinvention may find use in alemruzumab (Campath®, Millenium), a humanizedmonoclonal antibody currently approved for treatment of B-cell chroniclymphocytic leukemia. The Fc engineered constant region(s) of thepresent invention may find use in a variety of antibodies that aresubstantially similar to other clinical products and candidates,including but not limited to muromonab-CD3 (Orthoclone OKT3®), ananti-CD3 antibody developed by Ortho Biotech/Johnson & Johnson,ibritumomab tiuxetan (Zevalin®), an anti-CD20 antibody developed byIDEC/Schering AG, gemtuzumab ozogamicin (Mylotarg®), an anti-CD33 (p67protein) antibody developed by Celltech/Wyeth, alefacept (Amevive®), ananti-LFA-3 Fc fusion developed by Biogen), abciximab (ReoPro®),developed by Centocor/Lilly, basiliximab (Simulect®), developed byNovartis, palivizumab (Synagis®), developed by MedImmune, infliximab(Remicade®), an anti-TNFalpha antibody developed by Centocor, adalimumab(Humira®), an anti-TNFalpha antibody developed by Abbott, Humicade™, ananti-TNFalpha antibody developed by Celltech, etanercept (Enbrel®), ananti-TNFalpha Fc fusion developed by Immunex/Amgen, ABX-CBL, ananti-CD147 antibody being developed by Abgenix, ABX-IL8, an anti-IL8antibody being developed by Abgenix, ABX-MA1, an anti-MUC18 antibodybeing developed by Abgenix, Pemtumomab (R1549, 90Y-muHMFG1), ananti-MUC1 In development by Antisoma, Therex (R1550), an anti-MUC1antibody being developed by Antisoma, AngioMab (A81405), being developedby Antisoma, HuBC-1, being developed by Antisoma, Thioplatin (AS1407)being developed by Antisoma, Antegren® (natalizumab), ananti-alpha-4-beta-1 (VLA-4) and alpha-4-beta-7 antibody being developedby Biogen, VLA-1 mAb, an anti-VLA-1 integrin antibody being developed byBiogen, LTBR mAb, an anti-lymphotoxin beta receptor (LTBR) antibodybeing developed by Biogen, CAT-152, an anti-TGF-β2 antibody beingdeveloped by Cambridge Antibody Technology, J695, an anti-IL-12 antibodybeing developed by Cambridge Antibody Technology and Abbott, CAT-192, ananti-TGFβ1 antibody being developed by Cambridge Antibody Technology andGenzyme, CAT-213, an anti-Eotaxin1 antibody being developed by CambridgeAntibody Technology, LymphoStat-B™ an anti-Blys antibody being developedby Cambridge Antibody Technology and Human Genome Sciences Inc.,TRAIL-R1mAb, an anti-TRAIL-R1 antibody being developed by CambridgeAntibody Technology and Human Genome Sciences, Inc., Avastin™(bevacizumab, rhuMAb-VEGF), an anti-VEGF antibody being developed byGenentech, an anti-HER receptor family antibody being developed byGenentech, Anti-Tissue Factor (ATF), an anti-Tissue Factor antibodybeing developed by Genentech, Xolair™ (Omalizumab), an anti-IgE antibodybeing developed by Genentech, Raptiva™ (Efalizumab), an anti-CD11aantibody being developed by Genentech and Xoma, MLN-02 Antibody(formerly LDP-02), being developed by Genentech and MilleniumPharmaceuticals, HuMax CD4, an anti-CD4 antibody being developed byGenmab, HuMax-IL15, an anti-11.15 antibody being developed by Genmab andAmgen, HuMax-Inflam, being developed by Genmab and Medarex,HuMax-Cancer, an anti-Heparanase I antibody being developed by Genmaband Medarex and Oxford GeoSciences, HuMax-Lymphoma, being developed byGenmab and Amgen, HuMax-TAC, being developed by Genmab, IDEC-131, andanti-CD40L antibody being developed by IDEC Pharmaceuticals, IDEC-151(Clenoliximab), an anti-CD4 antibody being developed by IDECPharmaceuticals, IDEC-114, an anti-CD80 antibody being developed by IDECPharmaceuticals, IDEC-152, an anti-CD23 being developed by IDECPharmaceuticals, anti-macrophage migration factor (MIF) antibodies beingdeveloped by IDEC Pharmaceuticals, BEC2, an anti-idiotypic antibodybeing developed by Imclone, IMC-1C11, an anti-KDR antibody beingdeveloped by Imclone, DC101, an anti-flk-1 antibody being developed byImclone, anti-VE cadherin antibodies being developed by Imclone,CEA-Cide™ (labetuzumab), an anti-carcinoembryonic antigen (CEA) antibodybeing developed by Immunomedics, LymphoCide™ (Epratuzumab), an anti-CD22antibody being developed by Immunomedics, AFP-Cide, being developed byImmunomedics, MyelomaCide, being developed by Immunomedics, LkoCide,being developed by Immunomedics, ProstaCide, being developed byImmunomedics, MDX-010, an anti-CTLA4 antibody being developed byMedarex, MDX-060, an anti-CD30 antibody being developed by Medarex,MDX-070 being developed by Medarex, MDX-018 being developed by Medarex,Osidem™ (IDM-1), and anti-Her2 antibody being developed by Medarex andImmuno-Designed Molecules, HuMax™-CD4, an anti-CD4 antibody beingdeveloped by Medarex and Genmab, HuMax-IL15, an anti-IL15 antibody beingdeveloped by Medarex and Genmab, CNTO 148, an anti-TNFα antibody beingdeveloped by Medarex and Centocor/J&J, CNTO 1275, an anti-cytokineantibody being developed by Centocor/J&J, MOR101 and MOR102,anti-intercellular adhesion molecule-1 (ICAM-1) (CD54) antibodies beingdeveloped by MorphoSys, MOR201, an anti-fibroblast growth factorreceptor 3 (FGFR-3) antibody being developed by MorphoSys, Nuvion®(visilizumab), an anti-CD3 antibody being developed by Protein DesignLabs, HuZAF™, an anti-gamma interferon antibody being developed byProtein Design Labs, Anti-α5β1 Integrin, being developed by ProteinDesign Labs, anti-IL-12, being developed by Protein Design Labs, ING-1,an anti-Ep-CAM antibody being developed by Xoma, and MLN01, ananti-Beta2 integrin antibody being developed by Xoma, an pI-ADC antibodybeing developed by Seattle Genetics, all of the above-cited referencesin this paragraph are expressly incorporated herein by reference.

The carboxy-terminal portion of each chain defines a constant regionprimarily responsible for effector function. Kabat et al. collectednumerous primary sequences of the variable regions of heavy chains andlight chains. Based on the degree of conservation of the sequences, theyclassified individual primary sequences into the CDR and the frameworkand made a list thereof (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5thedition, NIH publication, No. 91-3242, E. A. Kabat et al., entirelyincorporated by reference).

In the IgG subclass of immunoglobulins, there are several immunoglobulindomains in the heavy chain. By “immunoglobulin (Ig) domain” herein ismeant a region of an immunoglobulin having a distinct tertiarystructure. Of interest in the present invention are the heavy chaindomains, including, the constant heavy (CH) domains and the hingedomains. In the context of IgG antibodies, the IgG isotypes each havethree CH regions. Accordingly, “CH” domains in the context of IgG are asfollows: “CH1” refers to positions 118-220 according to the EU index asin Kabat. “CH2” refers to positions 237-340 according to the EU index asin Kabat, and “CH3” refers to positions 341-447 according to the EUindex as in Kabat. As shown herein and described below, the variants ofthe present invention can include substitutions in one or more of the CHregions, as well as the hinge region, discussed below.

It should be noted that the sequences depicted herein start at the CH1region, position 118; the variable regions are not included except asnoted. For example, the first amino acid of SEQ ID NO: 2, whiledesignated as position “1” in the sequence listing, corresponds toposition 118 of the CH1 region, according to EU numbering.

Another type of Ig domain of the heavy chain is the hinge region. By“hinge” or “hinge region” or “antibody hinge region” or “immunoglobulinhinge region” herein is meant the flexible polypeptide comprising theamino acids between the first and second constant domains of anantibody. Structurally, the IgG CH1 domain ends at EU position 220, andthe IgG CH2 domain begins at residue EU position 237. Thus for IgG theantibody hinge is herein defined to include positions 221 (D221 in IgG1)to 236 (G236 in IgG1), wherein the numbering is according to the EUindex as in Kabat. In some embodiments, for example in the context of anFc region, the lower hinge is included, with the “lower hinge” generallyreferring to positions 226 or 230. As noted herein, Fc variant variantscan be made in the hinge region as well.

The light chain generally comprises two domains, the variable lightdomain (containing the light chain CDRs and together with the variableheavy domains forming the Fv region), and a constant light chain region(often referred to as CL or Cκ).

Another region of interest for additional substitutions, outlined below,is the Fc region. By “Fc” or “Fc region” or “Fc domain” as used hereinis meant the polypeptide comprising the constant region of an antibodyexcluding the first constant region immunoglobulin domain and in somecases, part of the hinge. Thus Fc refers to the last two constant regionimmunoglobulin domains of IgA, IgD, and IgG, the last three constantregion immunoglobulin domains of IgE and IgM, and the flexible hingeN-terminal to these domains. For IgA and IgM, Fc may include the Jchain. For IgG, the Fc domain comprises immunoglobulin domains Cγ2 andCγ3 (Cγ2 and Cγ3) and the lower hinge region between Cγ1 (Cγ1) and Cγ2(Cγ2). Although the boundaries of the Fc region may vary, the human IgGheavy chain Fc region is usually defined to include residues C226 orP230 to its carboxyl-terminus, wherein the numbering is according to theEU index as in Kabat. In some embodiments, as is more fully describedbelow, amino acid modifications are made to the Fc region, for exampleto alter binding to one or more FcγR receptors or to the FcRn receptor.

In some embodiments, the antibodies are full length. By “full lengthantibody” herein is meant the structure that constitutes the naturalbiological form of an antibody, including variable and constant regions,including one or more modifications as outlined herein.

Alternatively, the antibodies can be a variety of structures, including,but not limited to, antibody fragments, monoclonal antibodies,bispecific antibodies, minibodies, domain antibodies, syntheticantibodies (sometimes referred to herein as “antibody mimetics”),chimeric antibodies, humanized antibodies, antibody fusions (sometimesreferred to as “antibody conjugates”), and fragments of each,respectively.

In one embodiment, the antibody is an antibody fragment, as long as itcontains at least one constant domain which can be engineered. Specificantibody fragments include, but are not limited to, (i) the Fab fragmentconsisting of VL, VH, CL and CH1 domains, (ii) the Fd fragmentconsisting of the VH and CH1 domains, (iii) F(ab′)2 fragments, abivalent fragment comprising two linked Fab fragments (vii) single chainFv molecules (scFv), wherein a. VH domain and a VL domain are linked bya peptide linker which allows the two domains to associate to form anantigen binding site (Bird et al., 1988, Science 242:423-426, Huston etal., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:5879-5883, entirelyincorporated by reference), (iv) “diabodies” or “triabodies”,multivalent or multispecific fragments constructed by gene fusion(Tomlinson et. al., 2000, Methods Enzymol. 326:461-479; WO94/13804;Holliger et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:6444-6448, allentirely incorporated by reference).

Other antibody fragments that can be used include fragments that containone or more of the CH1, CH2, CH3, hinge and CL domains of the inventionthat have been engineered. For example, Fc fusions are fusions of the Fcregion (CH2 and CH3, optionally with the hinge region) fused to anotherprotein. A number of Fc fusions are known the art and can be improved bythe addition of the variants of the invention. In the present case,antibody fusions can be made comprising CH1; CH1, CH2 and CH3; CH2; CH3;CH2 and CH3; CH1 and CH3, any or all of which can be made optionallywith the hinge region, utilizing any combination of Fc variantsdescribed herein.

B. Chimeric and Humanized Antibodies

In some embodiments, the antibody can be a mixture from differentspecies, e.g. a chimeric antibody and/or a humanized antibody. Ingeneral, both “chimeric antibodies” and “humanized antibodies” refer toantibodies that combine regions from more than one species. For example,“chimeric antibodies” traditionally comprise variable region(s) from amouse (or rat, in some cases) and the constant region(s) from a human.“Humanized antibodies” generally refer to non-human antibodies that havehad the variable-domain framework regions swapped for sequences found inhuman antibodies. Generally, in a humanized antibody, the entireantibody, except the CDRs, is encoded by a polynucleotide of humanorigin or is identical to such an antibody except within its CDRs. TheCDRs, some or all of which are encoded by nucleic acids originating in anon-human organism, are grafted into the beta-sheet framework of a humanantibody variable region to create an antibody, the specificity of whichis determined by the engrafted CDRs. The creation of such antibodies isdescribed in, e.g., WO 92/11018, Jones, 1986, Nature 321:522-525,Verhoeyen et al., 1988, Science 239:1534-1536, all entirely incorporatedby reference. “Backmutation” of selected acceptor framework residues tothe corresponding donor residues is often required to regain affinitythat is lost in the initial grafted construct (U.S. Pat. No. 5,530,101;U.S. Pat. No. 5,585,089; U.S. Pat. No. 5,693,761; U.S. Pat. No.5,693,762; U.S. Pat. No. 6,180,370; U.S. Pat. No. 5,859,205; U.S. Pat.No. 5,821,337; U.S. Pat. No. 6,054,297; U.S. Pat. No. 6,407,213, allentirely incorporated by reference). The humanized antibody optimallyalso will comprise at least a portion of an immunoglobulin constantregion, typically that of a human immunoglobulin, and thus willtypically comprise a human Fc region. Humanized antibodies can also begenerated using mice with a genetically engineered immune system, Roqueet al., 2004, Biotechnol. Prog. 20:639-654, entirely incorporated byreference. A variety of techniques and methods for humanizing andreshaping non-human antibodies are well known in the art (See Tsurushita& Vasquez, 2004, Humanization of Monoclonal Antibodies, MolecularBiology of B Cells, 533-545, Elsevier Science (USA), and referencescited therein, all entirely incorporated by reference), Humanizationmethods include but are not limited to methods described in Jones etal., 1986, Nature 321:522-525; Riechmann et al., 1988; Nature332:323-329; Verhoeyen et al., 1988, Science, 239:1534-1536; Queen etal., 1989, Proc Natl Acad Sci, USA 86:10029-33; He et al., 1998, J.Immunol. 160: 1029-1035; Carter et al., 1992, Proc Natl Acad Sci USA89:4285-9, Presta et al., 1997, Cancer Res. 57(20):4593-9; Gorman etal., 1991, Proc. Natl. Acad. Sci. USA 88:4181-4185; O'Connor et al.,1998, Protein Eng 11:321-8, all entirely incorporated by reference,Humanization or other methods of reducing the immunogenicity of nonhumanantibody variable regions may include resurfacing methods, as describedfor example in Roguska et al., 1994, Proc. Natl. Acad. Sci. USA91:969-973, entirely incorporated by reference. In one embodiment, theparent antibody has been affinity matured, as is known in the art.Structure-based methods may be employed for humanization and affinitymaturation, for example as described in U.S. Ser. No. 11/004,590.Selection based methods may be employed to humanize and/or affinitymature antibody variable regions, including but not limited to methodsdescribed in Wu et al., 1999, J. Mol. Biol. 294:151-162; Baca et al.,1997, J. Biol. Chem. 272(16): 10678-10684; Rosok et al., 1996, J. Biol.Chem. 271(37): 22611-22618; Rader et al., 1998, Proc. Natl. Acad. Sci.USA 95: 8910-8915; Krauss et al., 2003, Protein Engineering16(10):753-759, all entirely incorporated by reference. Otherhumanization methods may involve the grafting of only parts of the CDRs,including but not limited to methods described in U.S. Ser. No.09/810,510; Tan et al., 2002, J. Immunol. 169:1119-1125; De Pascalis etal., 2002, J. Immunol. 169:3076-3084, all entirely incorporated byreference.

In one embodiment, the antibodies of the invention can be multispecificantibodies, and notably bispecific antibodies, also sometimes referredto as “diabodies”. These are antibodies that bind to two (or more)different antigens, or different epitopes on the same antigen. Diabodiescan be manufactured in a variety of ways known in the art (Holliger andWinter, 1993, Current Opinion Biotechnol. 4:446-449, entirelyincorporated by reference), e.g., prepared chemically or from hybridhybridomas. In some cases, multispecific (for example bispecific)antibodies are not preferred.

In one embodiment, the antibody is a minibody. Minibodies are minimizedantibody-like proteins comprising a scFv joined to a CH3 domain. Hu etal., 1996, Cancer Res. 56:3055-3061, entirely incorporated by reference.In the present instance, the CH3 domain can be engineered to improvebinding to FcRn and/or increase in vivo serum half-life. In some cases,the scFv can be joined to the Fc region, and may include some or theentire hinge region.

The antibodies of the present invention are generally isolated orrecombinant. “Isolated,” when used to describe the various polypeptidesdisclosed herein, means a polypeptide that has been identified andseparated and/or recovered from a cell or cell culture from which it wasexpressed. Ordinarily, an isolated polypeptide will be prepared by atleast one purification step. An “isolated antibody,” refers to anantibody which is substantially free of other antibodies havingdifferent antigenic specificities.

“Specific binding” or “specifically binds to” or is “specific for” aparticular antigen or an epitope means binding that is measurablydifferent from a non-specific interaction. Specific binding can bemeasured, for example, by determining binding of a molecule compared tobinding of a control molecule, which generally is a molecule of similarstructure that does not have binding activity. For example, specificbinding can be determined by competition with a control molecule that issimilar to the target.

Specific binding for a particular antigen or an epitope can beexhibited, for example, by an antibody having a KD for an antigen orepitope of at least about 10⁻⁴ M, at least about 10⁻⁵ M, at least about10⁻⁶ M, at least about 10⁻⁷ M, at least about 10⁻⁸ M, at least about10⁻⁹ M, alternatively at least about 10⁻¹⁰M, at least about 10⁻¹¹ M, atleast about 10⁻¹² M, or greater, where KD refers to a dissociation rateof a particular antibody-antigen interaction. Typically, an antibodythat specifically binds an antigen will have a KD that, is 20-, 50-,100-, 500-, 1000-, 5,000-, 10,000- or more times greater for a controlmolecule relative to the antigen or epitope.

Also, specific binding for a particular antigen or an epitope can beexhibited, for example, by an antibody having a KA or Ka for an antigenor epitope of at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- ormore times greater for the epitope relative to a control, where KA or Karefers to an association rate of a particular antibody-antigeninteraction.

C. Fc Variants

The present invention relates to the generation of Fc variants ofantibodies. As discussed above, by “Fc” or “Fc region” or “Fc domain” asused herein is meant the polypeptide comprising the constant region ofan antibody excluding the first constant region immunoglobulin domainand in some cases, part of the hinge. Thus Fc refers to the last twoconstant region immunoglobulin domains of IgA, IgD, and IgG, the lastthree constant region immunoglobulin domains of IgE and IgM, and theflexible hinge N-terminal to these domains. For IgA and IgM, Fc mayinclude the J chain. For IgG, the Fc domain comprises immunoglobulindomains Cγ2 and Cγ3 (Cγ2 and Cγ3) and the lower hinge region between Cγ1(Cγ1) and Cγ2 (Cγ2). Although the boundaries of the Fc region may vary,the human IgG heavy chain Fc region is usually defined to includeresidues C226 or P230 to its carboxyl-terminus, wherein the numbering isaccording to the EU index as in Kabat. In some embodiments, as is morefully described below, amino acid modifications are made to the Fcregion, for example to alter binding to one or more FcγR receptors or tothe FcRn receptor.

The present invention relates to the generation of Fc variants ofantibodies to form “Fc variant antibodies”. Fc variants are made byintroducing amino acid mutations into the parent molecule. “Mutations”in this context are usually amino acid substitutions, although as shownherein, deletions and insertions of amino acids can also be done andthus are defined as mutations.

The Fc variant antibodies of the invention show increased binding toFcRn and/or increased in vivo serum half-life. By “FcRn” or “neonatal FcReceptor” as used herein is meant a protein that binds the IgG antibodyFc region and is encoded at least in part by an FcRn gene. The FcRn maybe from any organism, including but not limited to humans, mice, rats,rabbits, and monkeys. As is known in the art, the functional FcRnprotein comprises two polypeptides, often referred to as the heavy chainand light chain. The light chain is beta-2-microglobulin and the heavychain is encoded by the FcRn gene. Unless other wise noted herein, FcRnor an FcRn protein refers to the complex of FcRn heavy chain withbeta-2-microglobulin. In some cases, the FcRn variants bind to the humanFcRn receptor, or it may be desirable to design variants that bind torodent or primate receptors in addition, to facilitate clinical trials.

A variety of such substitutions are known and described in U.S. Ser.Nos. 12/341,769; 10/672,280; 10/822,231; 11/124,620; 11/174,287;11/396,495; 11/538,406; 11/538,411; and 12/020,443, each of which isincorporated herein by reference in its entirety for all purposes and inparticular for all outlined substitutions.

In some embodiments, an Fc variant antibody can be engineered to includeany of the following substitutions, alone or in any combination; 436I,436V, 311I, 311V, 428L, 434S, 428L/434S, 259I, 308F, 259I/308F,259I/308F/428L, 307Q/434S, 434A, 434H, 250Q/428L, M252Y/S254T/T256E,307Q/434A, 307Q//380A/434A, and 308P/434A, 311I/434S, 311V/434S,434S/436I, 434S/436V. Numbering is EU as in Kabat, and it is understoodthat the substitution is non-native to the starting molecule. As hasbeen shown previously, these FcRn substitutions work in IgG1, IgG2 andIgG1/G2 hybrid backbones, and are specifically included for IgG3 andIgG4 backbones and derivatives of any IgG isoform as well.

In further embodiments, an Fc variant antibody can be engineered toinclude any of the following substitutions, alone or in any combination:248Y, 249G, 253H, 253N, 253L, 253T, 253V, 253Q, 253M, 253Y, 253F, 253W,255H, 255I, 255Q, 255E, 255F, 255L, 255S, 255G, 255W, 255P, 284D, 284E,285D, 285E, 286Q, 286F, 286D, 286E, 286G, 286L, 286P, 286Y, 286W, 2861,286V, 286R, 286K, 286H, 288N, 288H, 288Q, 288Y, 288F, 288W, 288L, 288I,307M, 307E, 307R, 307D, 307G, 3071, 307N, 307P, 307Q, 307S, 307V, 307Y,307K, 307H, 307L, 307F, 307W, 309W, 309R, 309K, 309D, 309E, 309F, 309H,309I, 309P, 309Q, 309L, 309Y, 309M, 312E, 312R, 312K, 312Y, 338R, 338H,378S, 378V, 378G, 426W, 426H, 426L, 426V, 426Y, 426M, 426I, 426F, 426T,428I, 428V, 433G, 436F, 438W, 4381T, 438N, 438S, 438G, 438Y, 438F, 4381,438D, 436I, 436V, and 436W, where the numbering is according to the EUIndex as in Kabat, and it is understood that the substitution isnon-native to the starting molecule. As has been shown previously, theseFcRn substitutions work in IgG1, IgG2 and IgG1/G2 hybrid backbones, andare specifically included for IgG3 and IgG4 backbones and derivatives ofany IgG isoform as well.

In some embodiments, the Fc variant of the invention includes at leasttwo modifications, wherein one of said modifications is the substitutionN434S, and the other of said modifications is a substitution selectedfrom one or more of the following: 248Y, 249G, 253H, 253N, 253L, 253T,253V, 253Q, 253M, 253Y, 253F, 253W, 255H, 255I, 255Q, 255E, 255F, 255L,255S, 255G, 255W, 255P, 284D, 284E, 285D, 285E, 286Q, 286F, 286D, 286E,286G, 286L, 286P, 286Y, 286W, 286I, 286V, 286R, 286K, 286H, 288N, 288H,288Q, 288Y, 288F, 288W, 288L, 288I, 307M, 307E, 307R, 307D, 307G, 307I,307N, 307P, 307Q, 307S, 307V, 307Y, 307K, 307H, 307L, 307F, 307W, 309W,309R, 309K, 309D, 309E, 309F, 309H, 309I, 309P, 309Q, 309L, 309Y, 309M,312E, 312R, 312K, 312Y, 338R, 338H, 378S, 378V, 378G, 426W, 426H, 426L,426V, 426Y, 426M, 426I, 426F, 426T, 428I, 428V, 433G, 436F, 438W, 438H,438N, 438S, 438G, 438Y, 438F, 438I, 438D, 436I, 436V, and 436W, wherethe numbering is according to the EU Index as in Kabat, and it isunderstood that the substitution is non-native to the starting molecule.As has been shown previously, these FcRn substitutions work in IgG1,IgG2 and IgG1/G2 hybrid backbones, and are specifically included forIgG3 and IgG4 backbones and derivatives of any IgG isoform as well.

In some embodiments, an Fc variant antibody can be engineered to includeany of the following substitutions, alone or in any combination: 378T,378E, 378I, 378L, 378M, 426G, 426A, 426Q, 426P, 426K, and 426R, wherethe numbering is according to the EU Index as in Kabat, and it isunderstood that the substitution is non-native to the starting molecule.As has been shown previously, these FcRn substitutions work in IgG1,IgG2 and IgG1/G2 hybrid backbones, and are specifically included forIgG3 and IgG4 backbones and derivatives of any IgG isoform as well.

In some embodiments, an Fc variant antibody can be engineered to includeany of the substitutions in any of the following positions alone or inany combination: 307, 311, 378, 426, 428, 434, and 4.36. In furtherembodiments, the substitutions are as follows, again alone or in anycombination: 307Q, 308P, 311I, 311V, 378E, 378F, 3781, 378L, 378M, 378R,378T, 378V, 378Y, 426A, 426G, 426K, 426L, 426P, 426Q, 426R, 426V, 428L,434S, 436I, and 436V. In still further embodiments, the Fc variantantibody is engineered to include any of the of the followingsubstitutions, alone or in any combination of the following or with anyother of the substitutions discussed herein: T307Q/N434S/Q311I,T307Q/N434S/Q311V, T307Q/N434S/A378V, T307Q/N434S/S426V,T307Q/N434S/Y436I, T307Q/N434S/Y436V, Q311I/N434S/A378V,Q311I/N434S/A378T, Q311I/N434S/A378I, Q311I/N434S/A378L,Q311I/N434S/S426V, Q311I/N434S/Y436L Q311I/N434S/Y436V, Q311V/S426V,Q311I/Y436I, Q311I/Y436V, Q311I/S426V/Y436I, Q311V/S426V/Y436V,Q311V/N434S/A378V, Q311V/N434S/S426V, Q311V/N434S/Y436I,Q311V/N434S/Y436V, S426V/N434S/A378V, S426V/N434S/A378T,S426V/N434S/A378I, S426V/N434S/A378L, S426V/N434S/Y436I,S426V/N434S/Y436V, N434S/Y436I/A378V, N434S/Y436I/A378T,N434S/Y436I/A378I, N434S/Y436I/A378L, N434S/Y436V/A378V,N434S/Y436V/A378T, N434S/Y436V/A378I, N434S/Y436V/A378L,N434S/Y436V/S426G, N434S/Y436V/S426A, N434S/Y436V/S426Q,N434S/Y436V/S426P, N434S/Y436V7S426L, M428L/N434S/T307Q,M428L/N434S/Q311I, M428L/N434S/Q311V, M428L/N434S/A378V,M428L/N434S/S426V, M428L/N434S/Y436I, M428L/N434S/Y436V, V308P/N434S,A37817N434S, A378I/N434S, A378L/N434S, A378E/N434S, A378M/N434S,A378F/N434S, A378Y/N434S, A378R/N434S, S426G/N434S, S426A/N434S,S426P/N434S, S426Q/N434S, S426K/N434S, S426R/N434S, T307Q/Q311I, andT307Q/Q311V, where the numbering is according to the EU Index as inKabat, and it is understood that the substitution is non-native to thestarting molecule. As has been shown previously, these FcRnsubstitutions work in IgG1, IgG2 and IgG1/G2 hybrid backbones, and arespecifically included for IgG3 and IgG4 backbones and derivatives of anyIgG isoform as well.

In some embodiments, an Fc variant antibody can be engineered to includeat least two of the following substitutions, alone or in anycombination: 248Y, 249G, 253H, 253N, 253L, 253T, 253V, 253Q, 253M, 253Y,253F, 253W, 255H, 255I, 255Q, 255E, 255F, 255L, 255S, 255G, 255W, 255P,284D, 284E, 285D, 285E, 286Q, 286F, 286D, 286E, 286G, 286L, 286P, 286Y,286W, 286I, 286V, 286R, 286K, 286H, 288N, 288H, 288Q, 288Y, 288F, 288W,288L, 288I, 307M, 307E, 307R, 307D, 307G, 307I, 307N, 307P, 307Q, 307S,307V, 307Y, 307K, 307H, 307L, 307F, 307W, 309W, 309R, 309K, 309D, 309E,309F, 309H, 309I, 309P, 309Q, 309L, 309Y, 309M, 312E, 312R, 312K, 312Y,338R, 338H, 378S, 378V, 378G, 426W, 426H, 426L, 426V, 426Y, 426M, 426I,426F, 426T, 428I, 428V, 433G, 436F, 438W, 438H, 438N, 438S, 438G, 438Y,438F, 438I, 438D, 436I, 436V, and 436W, where the numbering is accordingto the EU Index as in Kabat, and it is understood that the substitutionis non-native to the starting molecule. As has been shown previously,these FcRn substitutions work in IgG1, IgG2 and IgG1/G2 hybridbackbones, and are specifically included for IgG3 and IgG4 backbones andderivatives of any IgG isoform as well.

In other embodiments, no Fc variants are made in the variable region(s)of the antibodies. This is to be distinguished from affinity maturationsubstitutions in the variable region(s) that are made to increasebinding affinity of the antibody to its antigen. That is, an Fc variantin the variable region(s) is generally significantly “silent” withrespect to binding affinity.

III. Other Amino Acid Substitutions

As will be appreciated by those in the art, the Fc variant antibodies ofthe invention can contain additional amino acid substitutions inaddition to the substitutions discussed above.

In some embodiments, amino acid substitutions are imported from oneisotype into the Fc variant antibody.

In the hinge region (positions 233-236), changes can be made to increaseeffector function. That is, IgG2 has lowered effector function, and as aresult, amino acid substitutions at these positions from PVA(deletion)can be changed to ELLG, and an additional G327A variant generated aswell.

In the CH3 region, a mutation at position 384 can be made, for examplesubstituting a non-native serine.

Additional mutations that can be made include adding either N-terminalor C-terminal (depending on the structure of the antibody or fusionprotein) “tails” or sequences of one or more Fc amino acids; forexample, glutamic acids and aspartic acids can be added to the CH3O-terminus; generally, from 1 to 5 amino acids are added.

Properties of the Fc Variant Antibodies of the Invention

The Fc variant antibodies of the invention display increased binding toFcRn and/or increased in vivo serum half-life.

In addition, some variants herein are generated to increase stability.As noted herein, a number of properties of antibodies affect theclearance rate (e.g. stability for half-life) in vivo. In addition toantibody binding to the FcRn receptor, other factors that contribute toclearance and half-life are serum aggregation, enzymatic degradation inthe serum, inherent immunogenicity of the antibody leading to clearingby the immune system, antigen-mediated uptake, FcR (non-FcRn) mediateduptake and non-serum distribution (e.g. in different tissuecompartments).

IV. Optional and Additional Fc Engineering Fc Engineering

In addition to substitutions made to increase binding affinity to FcRnand/or increase serum half life, other substitutions can be made in theFc region, in general for altering binding to FcγR receptors.

By “Fc gamma receptor”, “FcγR” or “FcgammaR” as used herein is meant anymember of the family of proteins that bind the IgG antibody Fc regionand is encoded by an FcγR gene. In humans this family includes but isnot limited to FcγRI (CD64), including isoforms FcγRIa, FcγRIb, andFcγRIc; FcγRII (CD32), including isoforms FcγRIIa (including allotypesH131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), andFcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (includingallotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1and FcγRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65,entirely incorporated by reference), as well as any undiscovered humanFcγRs or FcγR isoforms or allotypes. An FcγR may be from any organism,including but not limited to humans, mice, rats, rabbits, and monkeys.Mouse FcγRs include but are not limited to FcγRI (CD64), FcγRII (CD32),FcγRIII-1 (CD16), and FcγRIII-2 (CD16-2), as well as any undiscoveredmouse FcγRs or FcγR isoforms or allotypes.

There are a number of useful Fc substitutions that can be made to alterbinding to one or more of the FcγR receptors. Substitutions that resultin increased binding as well as decreased binding can be useful. Forexample, it is known that increased binding to FcγRIIIa generallyresults in increased ADCC (antibody dependent cell-mediatedcytotoxicity; the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause lysis of the target cell. Similarly, decreasedbinding to FcγRIIb (an inhibitory receptor) can be beneficial as well insome circumstances. Amino acid substitutions that find use in thepresent invention include those listed in U.S. Ser. No. 11/124,620(particularly FIG. 41), U.S. Ser. Nos. 11/174,287, 11/396,495,11/538,406, all of which are expressly incorporated herein by referencein their entirety and specifically for the variants disclosed therein.Particular variants that find use include, but are not limited to, 236A,239D, 239E, 332E, 332D, 239D/332E, 267D, 267E, 328F, 267E/328F,236A/332E, 239D/332E/330Y, 239D, 332E/330L and 299T.

V. Other Antibody Modifications

In addition to substitutions made to increase binding affinity to FcRnand/or increase in vivo serum half life, additional antibodymodifications can be made, as described in further detail below.

Affinity Maturation

In some embodiments, one or more amino acid modifications are made inone or more of the CDRs of the antibody. In general, only 1 or 2 or3-amino acids are substituted in any single CDR, and generally no morethan from 4, 5, 6, 7, 8 9 or 10 changes are made within a set of CDRs.However, it should be appreciated that any combination of nosubstitutions, 1, 2 or 3 substitutions in any CDR can be independentlyand optionally combined with any other substitution.

In some cases, amino acid modifications in the CDRs are referred to as“affinity maturation”. An “affinity matured” antibody is one having oneor more alteration(s) in one or more CDRs which results in animprovement in the affinity of the antibody for antigen, compared to aparent antibody which does not possess those alteration(s). In somecases, although rare, it may be desirable to decrease the affinity of anantibody to its antigen, but this is generally not preferred.

Affinity maturation can be done to increase the binding affinity of theantibody for the antigen by at least about 10% to 50-100-150% or more,or from 1 to 5 fold as compared to the “parent” antibody. Preferredaffinity matured antibodies will have nanomolar or even picomolaraffinities for the target antigen. Affinity matured antibodies areproduced by known procedures. See, for example, Marks et al., 1992,Biotechnology 10:779-783 that describes affinity maturation by variableheavy chain (VH) and variable light chain (VL) domain shuffling. Randommutagenesis of CDR and/or framework residues is described in: Barbas, etal. 1994, Proc. Nat, Acad. Sci, USA 91:3809-3813; Shier et al., 1995,Gene 169:147-155; Yelton et al., 1995, J. Immunol. 155:1994-2004;Jackson et al., 1995, J. Immunol. 154(7):3310-9; and Hawkins et al.,1992, J. Mol. Biol. 226:889-896, for example.

Alternatively, amino acid modifications can be made in one or more ofthe CDRs of the antibodies of the invention that are “silent”, e.g. thatdo not significantly alter the affinity of the antibody for the antigen.These can be made for a number of reasons, including optimizingexpression (as can be done for the nucleic acids encoding the antibodiesof the invention).

Thus, included within the definition of the CDRs and antibodies of theinvention are variant CDRs and antibodies; that is, the antibodies ofthe invention can include amino acid modifications in one or more of theCDRs of Ab79 and Ab19. In addition, as outlined below, amino acidmodifications can also independently and optionally be made in anyregion outside the CDRs, including framework and constant regions.

ADC Modifications

In some embodiments, the Fc variant antibodies of the invention areconjugated with drugs to form antibody-drug conjugates (ADCs). Ingeneral, ADCs are used in oncology applications, where the use ofantibody-drug conjugates for the local delivery of cytotoxic orcytostatic agents allows for the targeted delivery of the drug moiety totumors, which can allow higher efficacy, lower toxicity, etc. Anoverview of this technology is provided in Ducry et al., BioconjugateChem., 21:5-13 (2010), Carter et al., Cancer J. 14(3):154 (2008) andSenter, Current Opin. Chem. Biol. 13:235-244 (2009), all of which arehereby incorporated by reference in their entirety

Thus the invention provides Fc variant antibodies conjugated to drugs.Generally, conjugation is done by covalent attachment to the antibody,as further described below, and generally relies on a linker, often apeptide linkage (which, as described below, may be designed to besensitive to cleavage by proteases at the target site or not). Inaddition, as described above, linkage of the linker-drug unit (LU-D) canbe done by attachment to cysteines within the antibody. As will beappreciated by those in the art, the number of drug moieties perantibody can change, depending on the conditions of the reaction, andcan vary from 1:1 to 10:1 drug:antibody. As will be appreciated by thosein the art, the actual number is an average.

Thus the invention provides Fc variant antibodies conjugated to drugs.As described below, the drug of the ADC can be any number of agents,including but not limited to cytotoxic agents such as chemotherapeuticagents, growth inhibitory agents, toxins (for example, an enzymaticallyactive toxin of bacterial, fungal, plant, or animal origin, or fragmentsthereof), or a radioactive isotope (that is, a radioconjugate) areprovided. In other embodiments, the invention further provides methodsof using the ADCs.

Drugs for use in the present invention include cytotoxic drugs,particularly those which are used for cancer therapy. Such drugsinclude, in general, DNA damaging agents, anti-metabolites, naturalproducts and their analogs. Exemplary classes of cytotoxic agentsinclude the enzyme inhibitors such as dihydrofolate reductaseinhibitors, and thymidylate synthase inhibitors, DNA intercalators, DNAcleavers, topoisomerase inhibitors, the anthracycline family of drugs,the vinca drugs, the mitomycins, the bleomycins, the cytotoxicnucleosides, the pteridine family of drugs, diynenes, thepodophyllotoxins, dolastatins, maytansinoids, differentiation inducers,and taxols.

Members of these classes include, for example, methotrexate,methopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine,cytosine arabinoside, melphalan, leurosine, leurosideine, actinomycin,daunorubicin, doxorubicin, mitomycin C, mitomycin A, caminomycin,aminopterin, tallysomycin, podophyllotoxin and podophyllotoxinderivatives such as etoposide or etoposide phosphate, vinblastine,vincristine, vindesine, taxanes including taxol, taxotere retinoic acid,butyric acid, N8-acetyl spermidine, camptothecin, calicheamicin,esperamicin, ene-diynes, duocarmycin A, duocarmycin SA, calicheamicin,camptothecin, maytansinoids (including DM1), monomethylauristatin E(MMAE), monomethylauristatin F (MMAF), and maytansinoids (DM4) and theiranalogues.

Toxins may be used as antibody-toxin conjugates and include bacterialtoxins such as diphtheria toxin, plant toxins such as ricin, smallmolecule toxins such as geldanamycin (Mandler et al (2000) J. Nat,Cancer Inst. 92(19): 1573-1581; Mandler et al (2000) Bioorganic & Med.Chem. Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem.13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl.Acad. Sci. USA 93:8618-862.3), and calicheamicin (Lode et al (1998)Cancer Res. 58:2928; Hinman et al (1993) Cancer Res. 53:3336-3342).Toxins may exert their cytotoxic and cytostatic effects by mechanismsincluding tubulin binding, DNA binding, or topoisomerase inhibition.

Conjugates of an Fc variant antibody and one or more small moleculetoxins,

such as a maytansinoids, dolastatins, auristatins, a trichothecene,calicheamicin, and CC1065, and the derivatives of these toxins that havetoxin activity, are contemplated.

Maytansinoids

Maytansine compounds suitable for use as maytansinoid drug moieties arewell known in the art, and can be isolated from natural sourcesaccording to known methods, produced using genetic engineeringtechniques (see Yu et al (2002) PNAS 99:7968-7973), or maytansinol andmaytansinol analogues prepared synthetically according to known methods.As described below, drugs may be modified by the incorporation of afunctionally active group such as a thiol or amine group for conjugationto the antibody.

Exemplary maytansinoid drug moieties include those having a modifiedaromatic ring, such as: C-19-dechloro (U.S. Pat. No. 4,256,746)(prepared by lithium aluminum hydride reduction of ansamytocin P2);C-20-hydroxy (or C-20-demethyl)+/−C-19-dechloro (U.S. Pat. Nos.4,361,650 and 4,307,016) (prepared by demethylation using Streptomycesor Actinomyces or dechlorination using LAH); and C-20-demethoxy,C-20-acyloxy (—OCOR), +/−dechloro (U.S. Pat. No. 4,294,757) (prepared byacylation using acyl chlorides) and those having modifications at otherpositions

Exemplary maytansinoid drag moieties also include those havingmodifications such as: C-9-SH (U.S. Pat. No. 4,424,219) (prepared by thereaction of maytansinol with H2S or P2S5);C-14-alkoxymethyl(demethoxy/CH2OR) (U.S. Pat. No. 4,331,598);C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2OAc) (U.S. Pat. No.4,450,254) prepared from Nocardia); C-15-hydroxy/acyloxy (U.S. Pat. No.4,364,866) (prepared by the conversion of maytansinol by Streptomyces);C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (isolated fromTrewia nudlflora); C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and4,322,348) (prepared by the demethylation of maytansinol byStreptomyces); and 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared by thetitanium trichloride/LAH reduction of maytansinol).

Of particular use are DM1 (disclosed in U.S. Pat. No. 5,208,020,incorporated by reference) and DM4 (disclosed in U.S. Pat. No.7,276,497, incorporated by reference). See also a number of additionalmaytansinoid derivatives and methods in 5,416,064, WO/01/24763,7,303,749, 7,601,354, U.S. Ser. No. 12/631,508, WO02/098883, 6,441,163,7,368,565, WO02/16368 and WO04/1033272, all of which are expresslyincorporated by reference in their entirety.

ADC's containing maytansinoids, methods of making same, and theirtherapeutic use are disclosed, for example, in U.S. Pat. Nos. 5,208,020;5,416,064; 6,441,163 and European Patent EP 0 425 235 B1, thedisclosures of which are hereby expressly incorporated by reference, Liuet al., Proc. Natl. Acad, Sci. USA 93:8618-8623 (1996) described ADCscomprising a maytansinoid designated DM1 linked to the monoclonalantibody C242 directed against human colorectal cancer. The conjugatewas found to be highly cytotoxic towards cultured colon cancer cells,and showed antitumor activity in an in vivo tumor growth assay.

Chari et al., Cancer Research 52:127-131 (1992) describe ADCs in which amaytansinoid was conjugated via a disulfide linker to the murineantibody A7 binding to an antigen on human colon cancer cell lines, orto another murine monoclonal antibody TA. 1 that binds the HER-2/neuoncogene. The cytotoxicity of the TA.1-maytansonoid conjugate was testedin vitro on the human breast cancer cell line SK-BR-3, which expresses3×105 HER-2 surface antigens per cell. The drug conjugate achieved adegree of cytotoxicity similar to the free maytansinoid drug, whichcould be increased by increasing the number of maytansinoid moleculesper antibody molecule. The A7-maytansinoid conjugate showed low systemiccytotoxicity in mice.

Auristatins and Dolastatins

In some embodiments, the ADC comprises an Fc variant antibody conjugatedto dolastatins or dolostatin peptidic analogs and derivatives, theauristatins (U.S. Pat. Nos. 5,635,483; 5,780,588). Dolastatins andauristatins have been shown to interfere with microtubule dynamics, GTPhydrolysis, and nuclear and cellular division (Woyke et al (2001)Antimicrob. Agents and Chemother. 45(12):3580-3584) and have anticancer(U.S. Pat. No. 5,663,149) and antifungal activity (Pettit et al (1998)Antimicrob. Agents Chemother, 42:2961-2965). The dolastatin orauristatin drug moiety may be attached to the antibody through the N(amino) terminus or the C (carboxyl) terminus of the peptidic drugmoiety (WO 02/088172).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties DE and DF, disclosed in “Senter etal, Proceedings of the American Association for Cancer Research, Volume45, Abstract Number 623, presented Mar. 28, 2004 and described in UnitedStates Patent Publication No. 2005/0238648, the disclosure of which isexpressly incorporated by reference in its entirety.

An exemplary auristatin embodiment is MMAE (shown in FIG. 10 wherein thewavy line indicates the covalent attachment to a linker (L) of anantibody drug conjugate; see U.S. Pat. No. 6,884,869 expresslyincorporated by reference in its entirety).

Another exemplary auristatin embodiment is MMAF, shown in FIG. 10wherein the wavy line indicates the covalent attachment to a linker (L)of an antibody drag conjugate (US 2005/0238649, 5,767,237 and 6,124,431,expressly incorporated by reference in their entirety):

Additional exemplary embodiments comprising MMAE or MMAF and variouslinker components (described further herein) have the followingstructures and abbreviations (wherein Ab means antibody and p is 1 toabout 8):

Typically, peptide-based drug moieties can be prepared by forming apeptide bond between two or more amino acids and/or peptide fragments.Such peptide bonds can be prepared, for example, according to the liquidphase synthesis method (see E. Schroder and K. Lubke, “The Peptides”,volume 1, pp 76-136, 1965, Academic Press) that is well known in thefield of peptide chemistry. The auristatin/dolastatin drug moieties maybe prepared according to the methods of: U.S. Pat. No. 5,635,483; U.S.Pat. No. 5,780,588; Pettit et al (1989) J. Am. Chem. Soc. 111:5463-5465;Pettit et al (1998) Anti-Cancer Drug Design 13:243-277; Pettit, G. R.,et al Synthesis, 1996, 719-725; Pettit et al (1996) J. Chem. Soc. PerkinTrans. 1 5:859-863; and Doronina (2003) Nat Biotechnol 21(7):778-784.

Calicheamicin

In other embodiments, the ADC comprises an antibody of the inventionconjugated to one or more calicheamicin molecules. For example, Mylotargis the first commercial ADC drug and utilizes calicheamicin γ1 as thepayload (see U.S. Pat. No. 4,970,198, incorporated by reference in itsentirety). Additional calicheamicin derivatives are described in U.S.Pat. Nos. 5,264,586, 5,384,412, 5,550,246, 5,739,116, 5,773,001,5,767,285 and 5,877,296, all expressly incorporated by reference. Thecalicheamicin family of antibiotics are capable of producingdouble-stranded DNA breaks at sub-picomolar concentrations. For thepreparation of conjugates of the calicheamicin family, see U.S. Pat.Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710,5,773,001, 5,877,296 (all to American Cyanamid Company). Structuralanalogues of calicheamicin which may be used include, but are notlimited to, γ1I, α2I, α2I, N-acetyl-γ1I, PSAG and θI1 (Hinman et al.,Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research58:2925-2928 (1998) and the aforementioned U.S. patents to AmericanCyanamid). Another anti-tumor drug that the antibody can be conjugatedis QFA which is an antifolate. Both calicheamicin and QFA haveintracellular sites of action and do not readily cross the plasmamembrane. Therefore, cellular uptake of these agents through antibodymediated internalization greatly enhances their cytotoxic effects.

Duocarmycins

CC-1065 (see U.S. Pat. No. 4,169,888, incorporated by reference) andduocarmycins are members of a family of antitumor antibiotics utilizedin ADCs. These antibiotics appear to work through sequence-selectivelyalkylating DNA at the N3 of adenine in the minor groove, which initiatesa cascade of events that result in apoptosis.

Important members of the duocarmycins include duocarmycin A (U.S. Pat.No. 4,923,990, incorporated by reference) and duocarmycin SA (U.S. Pat.No. 5,101,038, incorporated by reference), and a large number ofanalogues as described in U.S. Pat. Nos. 7,517,903, 7,691,962,5,101,038; 5,641,780; 5,187,186; 5,070,092; 5,070,092; 5,641,780;5,101,038; 5,084,468, 5,475,092, 5,585,499, 5,846,545, WO2007/089149,WO2009/017394A1, 5,703,080, 6,989,452, 7,087,600, 7,129,261, 7,498,302,and 7,507,420, all of which are expressly incorporated by reference.

VI. Other Cytotoxic Agents

Other antitumor agents that can be conjugated to the antibodies of theinvention include BCNU, streptozoicin, vincristine and 5-fluorouracil,the family of agents known collectively LL-E33288 complex described inU.S. Pat. Nos. 5,053,394, 5,770,710, as well as esperamicins (U.S. Pat.No. 5,877,296).

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, cretin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present invention further contemplates an ADC formed between anantibody and a compound with nucleolytic activity (e.g., a ribonucleaseor a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of the tumor, the antibody may comprise ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radio-conjugated antibodies. Examples includeAt211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 andradioactive isotopes of Lit.

The radio- or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as Tc99m or I123, Re186, Re188 and In111 can be attached viaa cysteine residue in the peptide, Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al (1978) Biochem.Biophys. Res. Commun. 80: 49-57 can be used to incorporate Iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

For compositions comprising a plurality of antibodies, the drug loadingis represented by p, the average number of drag molecules per Antibody.Drag loading may range from 1 to 20 drugs (D) per Antibody. The averagenumber of drags per antibody in preparation of conjugation reactions maybe characterized by conventional means such as mass spectroscopy, ELISAassay, and HPLC. The quantitative distribution ofAntibody-Drug-Conjugates in terms of p may also be determined.

In some instances, separation, purification, and characterization ofhomogeneous Antibody-Drug-conjugates where p is a certain value fromAntibody-Drug-Conjugates with other drug loadings may be achieved bymeans such as reverse phase HPLC or electrophoresis. In exemplaryembodiments, p is 2, 3, 4, 5, 6, 7, or 8 or a fraction thereof.

The generation of Antibody-drag conjugate compounds can be accomplishedby any technique known to the skilled artisan. Briefly, theAntibody-drug conjugate compounds can include an Fc variant antibody asthe Antibody unit, a drug, and optionally a linker that joins the dragand the binding agent.

A number of different reactions are available for covalent attachment ofdrags and/or linkers to binding agents. This is can be accomplished byreaction of the amino acid residues of the binding agent, for example,antibody molecule, including the amine groups of lysine, the freecarboxylic acid groups of glutamic and aspartic acid, the sulfhydrylgroups of cysteine and the various moieties of the aromatic amino acids.A commonly used non-specific methods of covalent attachment is thecarbodiimide reaction to link a carboxy (or amino) group of a compoundto amino (or carboxy) groups of the antibody. Additionally, bifunctionalagents such as dialdehydes or imidoesters have been used to link theamino group of a compound to amino groups of an antibody molecule.

Also available for attachment of drugs to binding agents is the Schiffbase reaction. This method involves the periodate oxidation of a drugthat contains glycol or hydroxy groups, thus forming an aldehyde whichis then reacted with the binding agent. Attachment occurs via formationof a. Schiff base with amino groups of the binding agent,Isothiocyanates can also be used as coupling agents for covalentlyattaching drugs to binding agents. Other techniques are known to theskilled artisan and within the scope of the present invention.

In some embodiments, an intermediate, which is the precursor of thelinker, is reacted with the drug under appropriate conditions. In otherembodiments, reactive groups are used on the drug and/or theintermediate. The product of the reaction between the drug and theintermediate, or the derivatized drag, is subsequently reacted with anFc variant antibody of the invention under appropriate conditions.

It will be understood that chemical modifications may also be made tothe desired compound in order to make reactions of that compound moreconvenient for purposes of preparing conjugates of the invention. Forexample a functional group e.g. amine, hydroxyl, or sulfhydryl, may beappended to the drug at a position which has minimal or an acceptableeffect on the activity or other properties of the drug

VII. Linker Units

Typically, the antibody-drag conjugate compounds comprise a Linker unitbetween the drug unit and the antibody unit. In some embodiments, thelinker is cleavable under intracellular or extracellular conditions,such that cleavage of the linker releases the drug unit from theantibody in the appropriate environment. For example, solid tumors thatsecrete certain proteases may serve as the target of the cleavablelinker; in other embodiments, it is the intracellular proteases that areutilized. In yet other embodiments, the linker unit is not cleavable andthe drug is released, for example, by antibody degradation in lysosomes.

In some embodiments, the linker is cleavable by a cleaving agent that ispresent in the intracellular environment (for example, within a lysosomeor endosome or caveolea). The linker can be, for example, a peptidyllinker that is cleaved by an intracellular peptidase or protease enzyme,including, but not limited to, a lysosomal or endosomal protease. Insome embodiments, the peptidyl linker is at least two amino acids longor at least three amino acids long or more.

Cleaving agents can include, without limitation, cathepsins B and D andplasmin, all of which are known to hydrolyze dipeptide drug derivativesresulting in the release of active drug inside target cells (see, e.g.,Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). Peptidyllinkers that are cleavable by enzymes that are present inCD38-expressing cells. For example, a peptidyl linker that is cleavableby the thiol-dependent protease cathepsin-B, which is highly expressedin cancerous tissue, can be used (e.g., a Phe-Leu or a Gly-Phe-Leu-Glylinker (SEQ ID NO: 11). Other examples of such linkers are described,e.g., in U.S. Pat. No. 6,214,345, incorporated herein by reference inits entirety and for all purposes.

In some embodiments, the peptidyl linker cleavable by an intracellularprotease is a Val-Cit linker or a Phe-Lys linker (see, e.g., U.S. Pat.No. 6,214,345, which describes the synthesis of doxorubicin with theval-cit linker).

In other embodiments, the cleavable linker is pH-sensitive, that is,sensitive to hydrolysis at certain pH values. Typically, thepH-sensitive linker hydrolyzable under acidic conditions. For example,an acid-labile linker that is hydrolyzable in the lysosome (for example,a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide,orthoester, acetal, ketal, or the like) may be used. (See, e.g., U.S.Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999,Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem.264:14653-14661.) Such linkers are relatively stable under neutral pHconditions, such as those in the blood, but are unstable at below pH 5.5or 5.0, the approximate pH of the lysosome. In certain embodiments, thehydrolyzable linker is a thioether linker (such as, e.g., a thioetherattached to the therapeutic agent via an acylhydrazone bond (see, e.g.,U.S. Pat. No. 5,622,929).

In yet other embodiments, the linker is cleavable under reducingconditions (for example, a disulfide linker). A variety of disulfidelinkers are known in the art, including, for example, those that can beformed using SATA (N-succinimidyl-5-acetylthioacetate), SPDP(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB(N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT(N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene)-,SPDB and SMPT. (See, e.g., Thorpe et al., 1987, Cancer Res.47:5924-5931; Wawrzynczak et al., In Immunoconjugates: AntibodyConjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed.,Oxford U. Press, 1987. See also U.S. Pat. No. 4,880,935.)

In other embodiments, the linker is a malonate linker (Johnson et al.,1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau etal., 1995, Bioorg-Med-Chem. 3(10): 1299-1304), or a 3′-N-amide analog(Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).

In yet other embodiments, the linker unit is not cleavable and the drugis released by antibody degradation. (See U.S. Publication No.2005/0238649 incorporated by reference herein in its entirety and forall purposes).

In many embodiments, the linker is self-immolative. As used herein, theterm “self-immolative Spacer” refers to a bifunctional chemical moietythat is capable of covalently linking together two spaced chemicalmoieties into a stable tripartite molecule. It will spontaneouslyseparate from the second chemical moiety if its bond to the first moietyis cleaved. See for example, WO 2007059404A2, WO06110476A2,WO05112919A2, WO2010/062171, WO09/017,394, WO07/089,149, WO 07/018,431,WO04/043493 and WO02/083180, which are directed to drug-cleavablesubstrate conjugates where the drug and cleavable substrate areoptionally linked through a self-immolative linker and which are allexpressly incorporated by reference.

Often the linker is not substantially sensitive to the extracellularenvironment. As used herein, “not substantially sensitive to theextracellular environment,” in the context of a linker, means that nomore than about 20%, 15%, 10%, 5%, 3%, or no more than about 1% of thelinkers, in a sample of antibody-drag conjugate compound, are cleavedwhen the antibody-drug conjugate compound presents in an extracellularenvironment (for example, in plasma).

Whether a linker is not substantially sensitive to the extracellularenvironment can be determined, for example, by incubating with plasmathe antibody-drug conjugate compound for a predetermined time period(for example, 2, 4, 8, 16, or 24 hours) and then quantitating the amountof free drug present in the plasma.

In other, non-mutually exclusive embodiments, the linker promotescellular internalization. In certain embodiments, the linker promotescellular internalization when conjugated to the therapeutic agent (thatis, in the milieu of the linker-therapeutic agent moiety of theantibody-drug conjugate compound as described herein). In yet otherembodiments, the linker promotes cellular internalization whenconjugated to both the auristatin compound and the Fc variant antibodiesof the invention.

A variety of exemplary linkers that can be used with the presentcompositions and methods are described in WO 2004-010957, U.S.Publication No. 2006/0074008, U.S. Publication No. 20050238649, and U.S.Publication No, 2006/0024317 (each of which is incorporated by referenceherein in its entirety and for all purposes).

VIII. Drug Loading

Drag loading is represented by p and is the average number of Dragmoieties per antibody in a molecule. Drug loading (“p”) may be 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or moremoieties (D) per antibody, although frequently the average number is afraction or a decimal. Generally, drug loading of from 1 to 4 isfrequently useful, and from 1 to 2 is also useful. ADCs of the inventioninclude collections of antibodies conjugated with a range of dragmoieties, from 1 to 20. The average number of drug moieties per antibodyin preparations of ADC from conjugation reactions may be characterizedby conventional means such as mass spectroscopy and, ELISA assay.

The quantitative distribution of ADC in terms of p may also bedetermined. In some instances, separation, purification, andcharacterization of homogeneous ADC where p is a certain value from ADCwith other drug loadings may be achieved by means such aselectrophoresis.

For some antibody-drug conjugates, p may be limited by the number ofattachment sites on the antibody. For example, where the attachment is acysteine thiol, as in the exemplary embodiments above, an antibody mayhave only one or several cysteine thiol groups, or may have only one orseveral sufficiently reactive thiol groups through which a linker may beattached. In certain embodiments, higher drug loading, e.g. p>5, maycause aggregation, insolubility, toxicity, or loss of cellularpermeability of certain antibody-drug conjugates. In certainembodiments, the drug loading for an ADC of the invention ranges from 1to about 8; from about 2 to about 6; from about 3 to about 5; from about3 to about 4; from about 3.1 to about 3.9; from about 3.2 to about 3.8;from about 3.2 to about 3.7; from about 3.2 to about 3.6; from about 3.3to about 3.8; or from about 3.3 to about 3.7. Indeed, it has been shownthat for certain ADCs, the optimal ratio of drug moieties per antibodymay be less than 8, and may be about 2 to about 5. See US 2005-0238649A1 (herein incorporated by reference in its entirety).

In certain embodiments, fewer than the theoretical maximum of drugmoieties are conjugated to an antibody during a conjugation reaction. Anantibody may contain, for example, lysine residues that do not reactwith the drug-linker intermediate or linker reagent, as discussed below.Generally, antibodies do not contain many free and reactive cysteinethiol groups which may be linked to a drug moiety; indeed most cysteinethiol residues in antibodies exist as disulfide bridges. In certainembodiments, an antibody may be reduced with a reducing agent such asdithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partialor total reducing conditions, to generate reactive cysteine thiolgroups. In certain embodiments, an antibody is subjected to denaturingconditions to reveal reactive nucleophilic groups such as lysine orcysteine.

The loading (drug/antibody ratio) of an ADC may be controlled indifferent ways, e.g., by: (i) limiting the molar excess of drug-linkerintermediate or linker reagent relative to antibody, (ii) limiting theconjugation reaction time or temperature, (iii) partial or limitingreductive conditions for cysteine thiol modification, (iv) engineeringby recombinant techniques the amino acid sequence of the antibody suchthat the number and position of cysteine residues is modified forcontrol of the number and/or position of linker-drug attachments (suchas thioMab or fhioFab prepared as disclosed herein and in WO2006/034488(herein incorporated by reference in its entirety)).

It is to be understood that where more than one nucleophilic groupreacts with a drug-linker intermediate or linker reagent followed bydrug moiety reagent, then the resulting product is a mixture of ADCcompounds with a distribution of one or more drag moieties attached toan antibody. The average number of drugs per antibody may be calculatedfrom the mixture by a dual ELISA antibody assay, which is specific forantibody and specific for the drug. Individual ADC molecules may beidentified in the mixture by mass spectroscopy and separated by HPLC,e.g. hydrophobic interaction chromatography.

In some embodiments, a homogeneous ADC with a single loading value maybe isolated from the conjugation mixture by electrophoresis orchromatography.

Methods of Determining Cytotoxic Effect of ADCs

Methods of determining whether a Drug or Antibody-Drug conjugate exertsa cytostatic and/or cytotoxic effect on a cell are known. Generally, thecytotoxic or cytostatic activity of an Antibody Drag conjugate can bemeasured by: exposing mammalian cells expressing a target protein of theAntibody Drag conjugate in a cell culture medium; culturing the cellsfor a period from about 6 hours to about 5 days; and measuring cellviability. Cell-based in vitro assays can be used to measure viability(proliferation), cytotoxicity, and induction of apoptosis (caspaseactivation) of the Antibody Drag conjugate.

For determining whether an Antibody Drag conjugate exerts a cytostaticeffect, a thymidine incorporation assay may be used. For example, cancercells expressing a target antigen at a density of 5,000 cells/well of a96-well plated can be cultured for a 72-hour period and exposed to 0.5μCi of ³H-thymidine during the final 8 hours of the 72-hour period. Theincorporation of ³H-thymidine into cells of the culture is measured inthe presence and absence of the Antibody Drug conjugate.

For determining cytotoxicity, necrosis or apoptosis (programmed celldeath) can be measured. Necrosis is typically accompanied by increasedpermeability of the plasma membrane; swelling of the cell, and ruptureof the plasma membrane. Apoptosis is typically characterized by membraneblebbing, condensation of cytoplasm, and the activation of endogenousendonucleases. Determination of any of these effects on cancer cellsindicates that an Antibody Drug conjugate is useful in the treatment ofcancers.

Cell viability can be measured by determining in a cell the uptake of adye such as neutral red, trypan blue, or ALAMAR™ blue (see, e.g., Pageet al., 1993, Intl. J. Oncology 3:473-476). In such an assay, the cellsare incubated in media containing the dye, the cells are washed, and theremaining dye, reflecting cellular uptake of the dye, is measuredspectrophotometrically. The protein-binding dye sulforhodamine B (SRB)can also be used to measure cytoxicity (Skehan et al., 1990, J. NatlCancer Inst. 82:1107-12).

Alternatively, a tetrazolium salt, such as MTT, is used in aquantitative colorimetric assay for mammalian cell survival andproliferation by detecting living, but not dead, cells (see, e.g.,Mosmann, 1983, J. Immunol. Methods 65:55-63).

Apoptosis can be quantitated by measuring, for example, DNAfragmentation. Commercial photometric methods for the quantitative invitro determination of DNA fragmentation are available. Examples of suchassays, including TUNEL (which detects incorporation of labelednucleotides in fragmented DNA) and ELISA-based assays, are described inBiochemica, 1999, no. 2, pp. 34-37 (Roche Molecular Biochemicals).

Apoptosis can also be determined by measuring morphological changes in acell. For example, as with necrosis, loss of plasma membrane integritycan be determined by measuring uptake of certain dyes (e.g., afluorescent dye such as, for example, acridine orange or ethidiumbromide). A method for measuring apoptotic cell number has beendescribed by Duke and Cohen, Current Protocols in Immunology (Coligan etal. eds., 1992, pp. 3.17.1-3.17.16). Cells also can be labeled with aDNA dye (e.g., acridine orange, ethidium bromide, or propidium iodide)and the cells observed for chromatin condensation and margination alongthe inner nuclear membrane. Other morphological changes that can bemeasured to determine apoptosis include, e.g., cytoplasmic condensation,increased membrane blebbing, and cellular shrinkage.

The presence of apoptotic cells can be measured in both the attached and“floating” compartments of the cultures. For example, both compartmentscan be collected by removing the supernatant, trypsinizing the attachedcells, combining the preparations following a centrifugation wash step(e.g., 10 minutes at 2000 rpm), and detecting apoptosis (e.g., bymeasuring DNA fragmentation). (See, e.g., Piazza et al., 1995, CancerResearch 55:3110-16).

In vivo, the effect of a therapeutic composition of the Fc variantantibody of the invention can be evaluated in a suitable animal model.For example, xenogenic cancer models can be used, wherein cancerexplants or passaged xenograft tissues are introduced into immunecompromised animals, such as nude or SCID mice (Klein et al., 1997,Nature Medicine 3: 402-408). Efficacy can be measured using assays thatmeasure inhibition of tumor formation, tumor regression or metastasis,and the like.

The therapeutic compositions used in the practice of the foregoingmethods can be formulated into pharmaceutical compositions comprising acarrier suitable for the desired delivery method. Suitable carriersinclude any material that when combined with the therapeutic compositionretains the anti-tumor function of the therapeutic composition and isgenerally non-reactive with the patient's immune system. Examplesinclude, but are not limited to, any of a number of standardpharmaceutical carriers such as sterile phosphate buffered salinesolutions, bacteriostatic water, and the like (see, generally,Remington's Pharmaceutical Sciences 16th Edition, A. Osal., Ed., 1980).

Glycosylation

Another type of covalent modification is alterations in glycosylation.In another embodiment, the antibodies disclosed herein can be modifiedto include one or more engineered glycoforms. By “engineered glycoform”as used herein is meant a carbohydrate composition that is covalentlyattached to the antibody, wherein said carbohydrate composition differschemically from that of a parent antibody. Engineered glycoforms may beuseful for a variety of purposes, including but not limited to enhancingor reducing effector function. A preferred form of engineered glycoformis afucosylation, which has been shown to be correlated to an increasein ADCC function, presumably through tighter binding to the FcγRIIIareceptor. In this context, “afucosylation” means that the majority ofthe antibody produced in the host cells is substantially devoid offucose, e.g. 90-95-98% of the generated antibodies do not haveappreciable fucose as a component of the carbohydrate moiety of theantibody (generally attached at N297 in the Fc region). Definedfunctionally, afucosylated antibodies generally exhibit at least a 50%or higher affinity to the FcγRIIIa receptor.

Engineered glycoforms may be generated by a variety of methods known inthe art (Umaña et al., 1999, Nat Biotechnol 17:176-180; Davies et al.,2001, Biotechnol Bioeng 74:288-294; Shields et al., 2002, J Biol Chem277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473; U.S.Pat. No. 6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser. No. 10/113,929;PCT WO 00/61739A1; PCT WO 01/29246A1; PCT WO 02/31140A1; PCT WO02/30954A1, all entirely incorporated by reference; (Potelligent®technology [Biowa, Inc., Princeton, N.J.]; GlycoMAb® glycosylationengineering technology [Glycart Biotechnology AG, Zürich, Switzerland]).Many of these techniques are based on controlling the level offucosylated and/or bisecting oligosaccharides that are covalentlyattached to the Fc region, for example by expressing an IgG in variousorganisms or cell lines, engineered or otherwise (for example Lee-13 CHOcells or rat hybridoma YB2/0 cells, by regulating enzymes involved inthe glycosylation pathway (for example FUT8 [α1,6-fucosyltranserase]and/or β1-4-N-acetylglucosaminyltransferase III [GnTIII]), or bymodifying carbohydrate(s) after the IgG has been expressed. For example,the “sugar engineered antibody” or “SEA technology” of Seattle Geneticsfunctions by adding modified saccharides that inhibit fucosylationduring production; see for example 20090317869, hereby incorporated byreference in its entirety. Engineered glycoform typically refers to thedifferent carbohydrate or oligosaccharide; thus an antibody can includean engineered glycoform.

Alternatively, engineered glycoform may refer to the IgG variant thatcomprises the different carbohydrate or oligosaccharide. As is known inthe art, glycosylation patterns can depend on both the sequence of theprotein (e.g., the presence or absence of particular glycosylation aminoacid residues, discussed below), or the host cell or organism in whichthe protein is produced. Particular expression systems are discussedbelow.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tri-peptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tri-peptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid,most commonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tri-peptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thestarting sequence (for O-linked glycosylation sites). For ease, theantibody amino acid sequence is preferably altered through changes atthe DNA level, particularly by mutating the DNA encoding the targetpolypeptide at preselected bases such that codons are generated thatwill translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on theantibody is by chemical or enzymatic coupling of glycosides to theprotein. These procedures are advantageous in that they do not requireproduction of the protein in a host cell that has glycosylationcapabilities for N- and O-linked glycosylation. Depending on thecoupling mode used, the sugar(s) may be attached to (a) arginine andhistidine, (b) free carboxyl groups, (c) free sulfhydryl groups such asthose of cysteine, (d) free hydroxyl groups such as those of serine,threonine, or hydroxy-proline, (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan, or (f) the amide group ofglutamine. These methods are described in WO 87/05330 and in Aplin andWriston, 1981, CRC Crit. Rev. Biochem., pp. 259-306, both entirelyincorporated by reference.

Removal of carbohydrate moieties present on the starting antibody (e.g.post-translationally) may be accomplished chemically or enzymatically.Chemical deglycosylation requires exposure of the protein to thecompound tritfluoromethanesulfonic acid, or an equivalent compound. Thistreatment results in the cleavage of most or all sugars except thelinking sugar (N-acetylglucosamine or N-acetylgalactosamine), whileleaving the polypeptide intact. Chemical deglycosylation is described byHakimuddin et al., 1987, Arch. Biochem. Biophys. 259:52 and by Edge etal., 1981, Anal, Biochem. 118:131, both entirely incorporated byreference. Enzymatic cleavage of carbohydrate moieties on polypeptidescan be achieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al., 1987, Meth. Enzymol. 138:350, entirelyincorporated by reference. Glycosylation at potential glycosylationsites may be prevented by the use of the compound tunicamycin asdescribed by Duskin et al., 1982, J, Biol, Chem, 257:3105, entirelyincorporated by reference, Tunicamycin blocks the formation ofprotein-N-glycoside linkages.

Another type of covalent modification of the antibody comprises linkingthe antibody to various nonproteinaceous polymers, including, but notlimited to, various polyols such as polyethylene glycol, polypropyleneglycol or polyoxyalkylenes, in the manner set forth in, for example,2005-2006 PEG Catalog from Nektar Therapeutics (available at the Nektarwebsite) U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144; 4,670,417;4,791,192 or 4,179,337, all entirely incorporated by reference. Inaddition, as is known in the art, amino acid substitutions may be madein various positions within the antibody to facilitate the addition ofpolymers such as PEG. See for example, U.S. Publication No.2005/0114037A1, entirely incorporated by reference.

IX. Nucleic Acids and Host Cells

Included within the invention are the nucleic acids encoding the Fcvariant antibodies of the invention. In the case where both a heavy andlight chain constant domains are included in the Fc variant antibody,generally these are made using nucleic acids encoding each, that arecombined into standard host cells (e.g. CHO cells, etc.) to produce thetetrameric structure of the antibody. If only one Fc variant engineeredconstant domain is being made, only a single nucleic acid will be used.

X. Antibody Compositions for In Vivo Administration

The use of the Fc variant antibodies of the invention in therapy willdepend on the antigen binding component; e.g. in the case of full lengthstandard therapeutic antibodies, on the antigen to which the antibody'sFv binds. That is, as will be appreciated by those in the art, thetreatment of specific diseases can be done with the additional benefitof increased half life of the molecule. This can result in a variety ofbenefits, including, but not limited to, less frequent dosing (which canlead to better patient compliance), lower dosing, and lower productioncosts.

Formulations of the antibodies used in accordance with the presentinvention are prepared for storage by mixing an antibody having thedesired degree of purity with optional pharmaceutically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. [1980]), in the form of lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, it may be desirable to provide antibodies with otherspecificities. Alternatively, or in addition, the composition maycomprise a cytotoxic agent, cytokine, growth inhibitory agent and/orsmall molecule antagonist. Such molecules are suitably present incombination in amounts that are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration should besterile, or nearly so. This is readily accomplished by filtrationthrough sterile filtration membranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and .gamma.ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods.

When encapsulated antibodies remain in the body for a long time, theymay denature or aggregate as a result of exposure to moisture at 37° C.,resulting in a loss of biological activity and possible changes inimmunogenicity. Rational strategies can be devised for stabilizationdepending on the mechanism involved. For example, if the aggregationmechanism is discovered to be intermolecular S—S bond formation throughthio-disulfide interchange, stabilization may be achieved by modifyingsulfhydryl residues, lyophilizing from acidic solutions, controllingmoisture content, using appropriate additives, and developing specificpolymer matrix compositions.

XI. Administrative Modalities

The antibodies and chemotherapeutic agents of the invention areadministered to a subject, in accord with known methods, such asintravenous administration as a bolus or by continuous infusion over aperiod of time, by intramuscular, intraperitoneal, intracerobrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes. Intravenous or subcutaneousadministration of the antibody is preferred,

XII. Treatment Modalities

In the methods of the invention, therapy is used to provide a positivetherapeutic response with respect to a disease or condition. By“positive therapeutic response” is intended an improvement in thedisease or condition, and/or an improvement in the symptoms associatedwith the disease or condition. For example, a positive therapeuticresponse would refer to one or more of the following improvements in thedisease: (1) a reduction in the number of neoplastic cells; (2) anincrease in neoplastic cell death; (3) inhibition of neoplastic cellsurvival; (5) inhibition (i.e., slowing to some extent, preferablyhalting) of tumor growth; (6) an increased patient survival rate; and(7) some relief from one or more symptoms associated with the disease orcondition.

Positive therapeutic responses in any given disease or condition can bedetermined by standardized response criteria specific to that disease orcondition. Tumor response can be assessed for changes in tumormorphology (i.e., overall tumor burden, tumor size, and the like) usingscreening techniques such as magnetic resonance imaging (MRI) scan,x-radiographic imaging, computed tomographic (CT) scan, bone scanimaging, endoscopy, and tumor biopsy sampling including bone marrowaspiration (BMA) and counting of tumor cells in the circulation.

In addition to these positive therapeutic responses, the subjectundergoing therapy may experience the beneficial effect of animprovement in the symptoms associated with the disease.

Thus for B cell tumors, the subject may experience a decrease in theso-called B symptoms, i.e., night sweats, fever, weight loss, and/orurticaria. For pre-malignant conditions, therapy with an Fc varianttherapeutic agent may block and/or prolong the time before developmentof a related malignant condition, for example, development of multiplemyeloma in subjects suffering from monoclonal gammopathy of undeterminedsignificance (MGUS).

An improvement in the disease may be characterized as a completeresponse. By “complete response” is intended an absence of clinicallydetectable disease with normalization of any previously abnormalradiographic studies, bone marrow, and cerebrospinal fluid (CSF) orabnormal monoclonal protein in the case of myeloma.

Such a response may persist for at least 4 to 8 weeks, or sometimes 6 to8 weeks, following treatment according to the methods of the invention.Alternatively, an improvement in the disease may be categorized as beinga partial response. By “partial response” is intended at least about a50% decrease in all measurable tumor burden (i.e., the number ofmalignant cells present in the subject, or the measured bulk of tumormasses or the quantity of abnormal monoclonal protein) in the absence ofnew lesions, which may persist for 4 to 8 weeks, or 6 to 8 weeks.

Treatment according to the present invention includes a “therapeuticallyeffective amount” of the medicaments used. A “therapeutically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve a desired therapeutic result.

A therapeutically effective amount may vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability of the medicaments to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the antibody or antibody portion areoutweighed by the therapeutically beneficial effects.

A “therapeutically effective amount” for tumor therapy may also bemeasured by its ability to stabilize the progression of disease. Theability of a compound to inhibit cancer may be evaluated in an animalmodel system predictive of efficacy in human tumors.

Alternatively, this property of a composition may be evaluated byexamining the ability of the compound to inhibit cell growth or toinduce apoptosis by in vitro assays known to the skilled practitioner. Atherapeutically effective amount of a therapeutic compound may decreasetumor size, or otherwise ameliorate symptoms in a subject. One ofordinary skill in the art would be able to determine such amounts basedon such factors as the subject's size, the severity of the subject'ssymptoms, and the particular composition or route of administrationselected.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. Parenteral compositions may beformulated in dosage unit form for ease of administration and uniformityof dosage. Dosage unit form as used herein refers to physically discreteunits suited as unitary dosages for the subjects to be treated; eachunit contains a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

The specification for the dosage unit forms of the present invention aredictated by and directly dependent on (a) the unique characteristics ofthe active compound and the particular therapeutic effect to beachieved, and (b) the limitations inherent in the art of compoundingsuch an active compound for the treatment of sensitivity in individuals.

The efficient dosages and the dosage regimens for the Fc variantantibodies used in the present invention depend on the disease orcondition to be treated and may be determined by the persons skilled inthe art.

An exemplary, non-limiting range for a therapeutically effective amountof an Fc variant antibody used in the present invention is about 0,1-100 mg/kg, such as about 0.1-50 mg/kg, for example about 0.1-20 mg/kg,such as about 0.1-10 mg/kg, for instance about 0.5, about such as 0.3,about 1, or about 3 mg/kg. In another embodiment, he antibody isadministered in a dose of 1 mg/kg or more, such as a dose of from 1 to20 mg/kg, e.g. a dose of from 5 to 20 mg/kg, e.g. a dose of 8 mg/kg.

A medical professional having ordinary skill in the art may readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, a physician or a veterinarian couldstart doses of the medicament employed in the pharmaceutical compositionat levels lower than that required in order to achieve the desiredtherapeutic effect and gradually increase the dosage until the desiredeffect is achieved.

In one embodiment, the Fc variant antibody is administered by infusionin a weekly dosage of from 10 to 500 mg/kg such as of from 200 to 400mg/kg Such administration may be repeated, e.g., 1 to 8 times, such as 3to 5 times. The administration may be performed by continuous infusionover a period of from 2 to 24 hours, such as of from 2 to 12 hours.

In one embodiment, the Fc variant antibody is administered by slowcontinuous infusion over a long period, such as more than 24 hours, ifrequired to reduce side effects including toxicity.

In one embodiment the Fc variant antibody is administered in a weeklydosage of from 250 mg to 2000 mg, such as for example 300 mg, 500 mg,700 mg, 1000 mg, 1500 mg or 2000 mg, for up to 8 times, such as from 4to 6 times. The administration may be performed by continuous infusionover a period of from 2 to 24 hours, such as of from 2 to 12 hours. Suchregimen may be repeated one or more times as necessary, for example,after 6 months or 12 months. The dosage may be determined or adjusted bymeasuring the amount of compound of the present invention in the bloodupon administration by for instance taking out a biological sample andusing anti-idiotypic antibodies which target the antigen binding regionof the Fc variant antibody.

In a further embodiment, the Fc variant antibody is administered onceweekly for 2 to 12 weeks, such as for 3 to 10 weeks, such as for 4 to 8weeks.

In one embodiment, the Fc variant antibody is administered bymaintenance therapy, such as, e.g., once a week for a period of 6 monthsor more.

In one embodiment, the Fc variant antibody is administered by a regimenincluding one infusion of an Fc variant antibody followed by an infusionof an Fc variant antibody conjugated to a radioisotope. The regimen maybe repeated, e.g., 7 to 9 days later.

As non-limiting examples, treatment according to the present inventionmay be provided as a daily dosage of an antibody in an amount of about0.1-100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on atleast one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, or 40, or alternatively, at least one of week 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 afterinitiation of treatment, or any combination thereof, using single ordivided doses of every 24, 12, 8, 6, 4, or 2 hours, or any combinationthereof.

In some embodiments the Fc variant antibody molecule thereof is used incombination with one or more additional therapeutic agents, e.g. achemotherapeutic agent. Non-limiting examples of DNA damagingchemotherapeutic agents include topoisomerase I inhibitors (e.g.,irinotecan, topotecan, camptothecin and analogs or metabolites thereof,and doxorubicin); topoisomerase II inhibitors (e.g., etoposide,teniposide, and daunorubicin); alkylating agents (e.g., melphalan,chlorambucil, busulfan, thiotepa, ifosfamide, carmustine, lomustine,semustine, streptozocin, decarbazine, methotrexate, mitomycin C, andcyclophosphamide); DNA intercalators (e.g., cisplatin, oxaliplatin, andcarboplatin); DNA intercalators and free radical generators such asbleomycin; and nucleoside mimetics (e.g., 5-fluorouracil, capecitibine,gemcitabine, fludarabine, cytarabine, mercaptopurine, thioguanine,pentostatin, and hydroxyurea).

Chemotherapeutic agents that disrupt cell replication include:paclitaxel, docetaxel, and related analogs; vincristine, vinblastin, andrelated analogs; thalidomide, lenalidomide, and related analogs (e.g.,CC-5013 and CC-4047); protein tyrosine kinase inhibitors (e.g., imatinibmesylate and gefitinib); proteasome inhibitors (e.g., bortezomib); NF-κBinhibitors, including inhibitors of IκB kinase; antibodies which bind toproteins overexpressed in cancers and thereby downregulate cellreplication (e.g., trastuzumab, rituximab, cetuximab, and bevacizumab);and other inhibitors of proteins or enzymes known to be upregulated,over-expressed or activated in cancers, the inhibition of whichdownregulates cell replication.

In some embodiments, the antibodies of the invention can be used priorto, concurrent with, or after treatment with Velcade® (bortezomib).

EXAMPLES

Examples are provided below to illustrate the present invention. Theseexamples are not meant to constrain the present invention to anyparticular application or theory of operation. For all constant regionpositions discussed in the present invention, numbering is according tothe EU index as in Kabat (Kabat et al., 1991, Sequences of Proteins ofImmunological Interest, 5th Ed., United States Public Health Service,National Institutes of Health, Bethesda, entirely incorporated byreference). Those skilled in the art of antibodies will appreciate thatthis convention consists of nonsequential numbering in specific regionsof an immunoglobulin sequence, enabling a normalized reference toconserved positions in immunoglobulin families. Accordingly, thepositions of any given immunoglobulin as defined by the EU index willnot necessarily correspond to its sequential sequence.

Example 1 DNA Construction, Expression, and Purification of Fc Variants

Amino acid modifications were engineered in the Fc region of IgGantibodies to improve their affinity for the neonatoal Fc receptor FcRn.Variants were screened in the context of a number of different human IgGconstant chains (FIG. 2), including IgG1, IgG2, and a hybrid IgGsequences that contains the CH1 and upper hinge of IgG1 and the Fcregion of IgG2. It will be appreciated by those skilled in the art thatbecause of the different interactions of the IgG1 and IgG2 Fc regionwith FcγRs and complement, these different parent Fc regions will havedifferent FcγR− and complement-mediated effector function properties.Exemplary sequences of Fc variants in the context of these parent IgGconstant chains are shown in FIG. 3.

Fc variants were engineered in the context of an antibody targetingvascular endothelial factor (VEGF). The heavy and light chain variableregions (VH and VL) are those of a humanized version of the antibodyA4.6.1, also referred to as bevacizumab (Avastin®), which is approvedfor the treatment of a variety of cancers. The amino acid sequences ofthe VH and VL regions of this antibody are shown in FIG. 4.

Genes encoding the heavy and light chains of the anti-VEGF antibodieswere constructed in the mammalian expression vector pTT5. Human IgG1 andIgG2 constant chain genes were obtained from IMAGE clones and subclonedinto the pTT5 vector. The IgG1/2 gene was constructed using PCRmutagenesis. VH and VL genes encoding the anti-VEGF antibodies weresynthesized commercially (Blue Heron Biotechnologies, Bothell Wash.),and subcloned into the vectors encoding the appropriate CL, IgG1, IgG2,and IgG1/2 constant chains, Amino acid modifications were constructedusing site-directed mutagenesis using the QuikChange® site-directedmutagenesis methods (Stratagene, La Jolla Calif.). All DNA was sequencedto confirm the fidelity of the sequences.

Plasmids containing heavy chain gene (VH—Cγ1-Cγ2-Cγ3) wereco-transfected with plasmid containing light chain gene (VL-Cκ) into293E cells using lipofectamine (Invitrogen, Carlsbad Calif.) and grownin FreeStyle 293 media (Invitrogen, Carlsbad Calif.). After 5 days ofgrowth, the antibodies were purified from the culture supernatant byprotein A affinity using the MabSelect resin (GE Healthcare), Antibodyconcentrations were determined by bicinchoninic acid (BCA) assay(Pierce).

Example 2 Fc Variant Antibodies Maintain Binding to Antigen

The fidelity of the expressed variant antibodies was confirmed bydemonstrating that they maintained specificity for antigen, VEGF bindingwas monitored using surface plasmon resonance (SPR, Biacore), performedusing a Biacore 3000 instrument. Recombinant VEGF (VEGF-165, PeproTech,Rocky Hill, N.J.) was adhered to a CM5 chip surface by coupling withN-hydroxysuccinimide/N-ethyl-N′-(-3-dimethylamino-propyl) carbodiimide(NHS/EDC) using standard methods. WT and variant antibodies wereinjected as analytes, and response, measured in resonance units (RU),was acquired. The dissociation phase was too slow to measure trueequilibrium constants, and so relative binding was determined bymeasuring RU's at the end of the association phase, which should beproportional to the protein concentration (which is held constant in theexperiment) and the association rate constant. The data (FIG. 5) showthat the variant anti-VEGF antibodies maintain binding to antigen, incontrast to the negative control anti-Her2 antibody which does not bindVEGF.

Example 3 Measurement of Binding to Human FcRn

Binding of variant antibodies to human FcRn was measured at pH 6.0, thepH at which it is naturally bound in endosomes. Vectors encoding beta 2microglobulin and His-tagged alpha chain genes of FcRn were constructed,co-transfected into 293T cells, and purified using nickelchromatography. Antibody affinity for human FcRn (hFcRn) at pH 6.0 wasmeasured on a Biacore 3000 instrument by coupling human FcRn to a CM5chip surface using standard NHS/EDC chemistry. WT and variant antibodieswere used in the mobile phase at 25-100 nM concentration and responsewas measured in resonance units. Association and dissocation phases atpH 6.0 were acquired, followed by an injection of pH 7.4 buffer tomeasure release of antibody from receptor at the higher pH. A cycle withantibody and buffer only provided baseline response, which wassubtracted from each sample sensorgram.

FIG. 6 shows Biacore sensorgrams for binding of native IgG1 and selectFc variant antibodies to human FcRn at the two relevant pH's. The datashow that wild-type and variant antibodies bind readily to FcRn chip atpH 6.0 and dissociate slowly at that pH, as they would in the endosome,yet release rapidly at pH7.4, as they would upon recycling of endosometo the membrane and exposure to the higher pH of serum.

The FcRn association/dissociation curves did not fit to a simpleLangmuir model, possibly due to the antibody and receptor multi-valencyor chip heterogeneity. Pseudo-Ka values (referred to as Ka*) weredetermined by fitting to a conformational change model with the changein refractive index (RI) fixed at 0 RU. These values for select variantantibodies are plotted in FIG. 7. The relative affinity of each variantas compared to its parent IgG was calculated according to the equationFold=(WT Ka*/Variant Ka*).

Example 4 Pharmacokinetic Experiments in Human FcRn Knock-in Mice

To test the ability of select variants to improve half-life in vivo,pharmacokinetic experiments were performed in B6 mice that arehomozygous knock-outs for murine FcRn and heterozygous knock-ins ofhuman FcRn (mFcRn^(−/−), hFcRn⁺) (Petkova et al., 2006, Int. Immunol18(12): 1759-69, entirely incorporated by reference), herein referred toas hFcRn or hFcRn⁺ mice. A single, intravenous tail vein injection ofanti-VEGF antibody (2 mg/kg) was given to groups of 4-7 female micerandomized by body weight (20-30 g range). Blood (˜50 ul) was drawn fromthe orbital plexus at each time point, processed to serum, and stored at−80° C. until analysis. Study durations were 28 or 49 days.

Antibody concentrations were determined using two ELISA assays. In thefirst two studies (referred to as Study 1 and Study 2), goat anti-humanFc antibody (Jackson Immune research) was adhered to the plate, wellswere washed with PBST (phosphate buffered saline with 0.05% Tween) andblocked with 3% BSA in PBST. Serum or calibration standards were theadded, followed by PBST washing, addition of europium labeled anti-humanIgG (Perkin Elmer), and further PBST washing. The time resolvedfluorescence signal was collected. For Studies 3-5, serum concentrationwas detected using a similar ELISA, but recombinant VEGF (VEGF-165,PeproTech, Rocky Hill, N.J.) was used as capture reagent and detectionwas carried out with biotinylated anti-human kappa antibody andeuropium-labeled streptavidin. PK parameters were determined forindividual mice with a non-compartmental model using WinNonLin(Pharsight Inc, Mountain View Calif.). Nominal times and dose were usedwith uniform weighing of points. The time points used (lambda Z ranges)were from 4 days to the end of the study, although all time points wereused for the faster clearing mutants, P257N and P257L.

Five antibody PK studies in mFcRn^(−/−) hFcRn⁺ mice were carried out.FIG. 8 a-8 b shows serum concentration data for WT and variant IgG1(Study 3) and IgG2 (Study 5) antibodies respectively. Fitted PKparameters from all in vivo PK studies carried out in mFcRn^(−/−) hFcRn⁺mice are provided in FIG. 9, PK data include half-life, which representsthe beta phase that characterizes elimination of antibody from serum,Cmax, which represents the maximal observed serum concentration, AUG,which represents the area under the concetration time curve, andclearance, which represents the clearance of antibody from serum. Alsoprovided for each variant is the calculated fold improvement orreduction in half-life relative to the IgG1 or IgG2 parent antibody[Fold half-life=half-life(variant)/half-life (WT)].

Example 5 Pharmacokinetic Experiment in Nonhuman Primates

The PK properties of biologics in non-human primates arewell-established to be predictive of their properties in humans. A PKstudy was carried out in cynomolgus monkeys (macaca fascicularis) inorder to evaluate the capacity of the variant anti-VEGF antibodies toimprove serum half-life in non-human primates.

In preparation for a PK study in cynomolgus monkeys, binding of thevariant antibodies to cynomolgus (cyno) FcRn (cFcRn) at pH 6.0 wasmeasured. cFcRn was constructed, expressed, and purified as describedabove for human FcRn. Binding of variant anti-VEGF antibodies to cFcRnwas measured using Biacore as described above. The data is provided inFIG. 10 a-10 b. The results show that the variants improve affinity forcyno FcRn similarly as they do for human FcRn. Dissociation at thehigher pH (7.4) was also very rapid (data not shown), similar to as wasobserved for binding to human FcRn. These results are not surprisinggiven the high sequence homology of the human and cyno receptors (FcRnalpha chain 96%, beta-2-microglobulin 91%).

The PK of the variants were studied in vivo in non-human primates. Malecynomolgus monkeys (macaca fascicularis, also called crab-eatingMacaque) weighing 2.3-5.1 kg were randomized by weight and divided into5 groups with 3 monkeys per group. The monkeys were given a single, 1hour peripheral vein infusion of 4 mg/kg antibody. Blood samples (1 ml)were drawn from a separate vein from 5 minutes to 90 days aftercompletion of the infusion, processed to serum and stored at −70 C.Animals were not harmed during these studies.

Antibody concentrations were determined using the VEGF capture method asdescribed above. PK parameters were determined by fitting theconcentrations versus time to a non-compartmental model as was done inthe mouse PK studies. However, time points from day 10 to day 90 wereused for PK parameter determinations. The PK results are plotted in FIG.11, and the fitted parameters are provided in FIG. 12. The results showthat the variants enhanced the in vivo half-life of antibody up to3.2-fold. In the best case (the 428L/434S variant) half-life wasextended from 9.7 days to 31.1 days. The PK results obtained incynomolgus monkeys are consistent with those obtained in mFcRn^(−/−)hFcRn⁺ mice, validating the hFcRn mouse model as a system for assessingthe in vivo PK properties of the variants, and supporting theconclusions from those studies.

Example 6 Engineering Additional Fc Variants that Extend In VivoHalf-Life

New variants were engineered to further screen for modifications thatextend in vivo halt-life. Designed substitutions are shown in FIG. 13,Variants were constructed in the context of an antibody with thebevacizumab variant region and IgG1/2 N434S constant region; however,variants may be constructed in any IgG isotype. Variants wereconstructed, expressed, and purified as described above.

Variants were screened for binding to human FcRn at pH 6.0 by Biacore.Anti-VEGF antibodies were captured to a VEGF coupled chip, a singleconcentration of FcRn analyte was flowed over the chip (associationphase), and then buffer was washed over to dissociate FcRn analyte(dissociation phase). The dissociation off-rate (k_(off)) was determinedby fitting the dissociation phase data to a 1:1 Langmuir dissociationmodel. The results are shown numerically in FIG. 14, and plotted in FIG.15. As can be seen, a number of the designed variants improve FcRnbinding (reduce the k_(off)) relative to the IgG1/2 N434S background.

The FcRn binding of select variants was tested using the same Biacoreformat but with an analyte (FcRn) concentration series in order toobtain accurate equilibrium dissociation constants (K_(D)s). Sensorgramsat multiple analyte concentrations were fit globally to a 1:1 Langmuirbinding model. Resulting kinetic and equilibrium binding constants arepresented in FIG. 16, along with the Fold K_(D) and Fold k_(off)relative to IgG1 WT and IgG1/2 N434S respectively. Data are plotted inFIGS. 17 and 18. A number of variants, comprising several differentsubstitutions improve binding to human FcRn. Beneficial substitutionsinclude but are not limited to T307Q, Q311I, Q311V, A378V, S426V, S426T,Y436I, and Y436V. The successful engineering at positions 378 and 426 issurprising giving that they are more distal to the FcRn bindinginterface on Fc.

Additional substitutions were designed to explore whether othersubstitutions at these positions may provide improved binding to FcRn.These variants are listed in FIG. 19. Based on the collective data, anew library of variant combinations was designed to further screen forFc variants that have the potential to extend antibody half-life invivo. These variants are listed in FIG. 20.

Variants were constructed, expressed, and purified as described above.Variants were screened for binding to human FcRn at pH 6.0 by Biacore.Anti-VEGF antibodies were captured to a VEGF coupled chip, a singleconcentration of FcRn analyte was flowed over the chip (associationphase), and then buffer was washed over to dissociate FcRn analyte(dissociation phase). The dissociation off-rate (k_(off)) was determinedby fitting the dissociation phase data to a 1:1 Langmuir dissociationmodel. The results are shown numerically in FIG. 21, and plotted in FIG.22. As can be seen, a number of the designed variants improve FcRnbinding (reduce the k_(off)).

In vivo pharmacokinetic properties of the variants were tested in hFcRn⁺mice as described above. A single, intravenous tail vein injection ofanti-VEGF antibody (2 mg/kg) was given. Antibody concentrations weredetermined using the VEGF-capture ELISA assay described above. FIG. 23shows mean serum concentration data for Fc variant and IgG1/2 parentanti-VEGF antibodies. Fitted half-lives of individual mice and means areprovided in FIG. 24. FIG. 25 shows a scatter plot of the fittedhalf-lives from the individual mice in the study. The results indicatethat the engineered Fc variants extend half-life relative to the parentIgG1/2 antibody, as well as the IgG1/2 N434S variant.

Whereas particular embodiments of the invention have been describedabove for purposes of illustration, it will be appreciated by thoseskilled in the art that numerous variations of the details may be madewithout departing from the invention as described in the appendedclaims. All references cited herein are incorporated in their entirety.

1. An antibody comprising a variant Fc region as compared to a parent Fcregion, wherein said variant Fc region comprises a first mutation whichis a serine at position 434 and a second mutation selected from a groupconsisting of: an isoleucine at position 311, a valine at position 311,an isoleucine at position 436, and a valine at position 436, whereinsaid antibody has increased serum half-life as compared to an antibodycomprising said parent Fc region, and wherein numbering is according tothe ELI index.
 2. An antibody comprising a variant Fc region as comparedto a parent Fc region, wherein said variant Fc region comprises a firstmutation which is a serine at position 434 and a second mutationselected from a group consisting of: an isoleucine at position 311, avaline at position 311, an isoleucine at position 436, and a valine atposition 436, wherein said antibody has increased binding affinity to ahuman FcRn receptor as compared to an antibody comprising said parent Fcregion, and wherein numbering is according to the EU index.
 3. A nucleicacid encoding the variant Fc region of claim 1 or
 2. 4. A host cellcomprising the nucleic acid of claim
 21. 5. A method comprisingadministering an antibody to a subject, said method comprisingadministering an antibody comprising a variant Fc region as compared toa parent Fc region, wherein said variant Fc region comprises a firstmutation which is a serine at position 434 and a second mutationselected from a group consisting of: an isoleucine at position 311, avaline at position 311, an isoleucine at position 436, and a valine atposition 436, wherein said antibody has increased serum half-life ascompared to an antibody comprising said parent Fc region, and whereinnumbering is according to the EU index.